Salmonella strains for the treatment of cancer and use thereof

Abstract

This invention relates to a selectively cancer-targeting Salmonella strain for treating cancer, and compositions comprising said strain for the prevention or treatment of cancer. In particular, the Salmonella strain of this invention has tumor-inhibiting effects but exhibits significantly low viability in normal organs, thus potentially possessing significant anticancer activity compared to conventional inventions.

Description

TECHNICAL FIELD

[0001] The present application relates to a Salmonella strain selectively acting on cancer and a composition for preventing or treating cancer containing the same. BACKGROUND

[0002] Cancer is one of the diseases that causes the most deaths worldwide, and the incidence of cancer is increasing due to an increase in life expectancy and a decrease in the age of onset of cancer. According to the statistics provided by the National Cancer Center of Korea in 2013, the total number of Korean cancer patients registered at the Cancer Center of Korea in 2010 was 202,053, and there was a tendency to continue to increase.

[0003] Therefore, for cancer, research on cancer treatment has been conducted in all directions worldwide, starting from basic research at the cellular level. However, the mechanism of cancer development is not yet known, and it is difficult to prevent cancer recurrence and cure cancer, so the demand for anticancer drugs is growing explosively, and huge research funds are invested in research institutions and companies. However, the cost of cancer treatment is not only the direct medical cost, but also the indirect cost incurred due to the withdrawal from social and economic activities after the onset of cancer, recovery, and patient care, and it becomes a huge economic burden for all members of the family and society of cancer patients. Therefore, it is necessary to introduce a new low-cost technology.

[0004] According to this situation, since the report of the inhibition of cancer by bacteria about 100 years ago, many researchers have developed strains having an anticancer effect, and some research groups have been conducting clinical trials.

[0005] The cellular environment of cancer tissue is represented by low oxygen partial pressure, abundant nutrients, and lack of immune response, which can be a habitat characteristic preferred for the propagation of pathogenic strains in the body. In particular, the necrotic area located at the center of cancer tissue is known to be an area where drugs cannot be effectively delivered and cause cancer metastasis, etc. Clostridium, Bifidobacterium, Salmonella, etc. accumulate and propagate in the necrotic area located at the center of cancer tissue with abundant nutrients. In this process, cancer tissue contraction and growth inhibition are observed, and thus these strains are used as probiotic strains for treating cancer.

[0006] Dr. Xu's research group in China developed a system for delivering an angiogenesis inhibitor, endostatin, to solid cancer tissue by orally administering Bifidobacterium longum, and then reported a study on the treatment of cancer using a mixture containing doxorubicin, an anticancer antibiotic (Xu YF et al., Cancer Gene Ther., 2007, 14(2):1517; Hu B et al., Cancer Gene Ther., 2009, 16(8):655-63).

[0007] In addition, several research groups including the National Cancer Institute (NCI) reported the inhibitory effect of Salmonella on cancer growth and the anticancer effect of attenuated recombinant Salmonella loaded with anticancer therapeutic substances, including mutant strains obtained by attenuating the virulence of Salmonella typhimurium (Nishikawa H et al., J Clin Invest. 2006, 116(7): 1946-54, Clairmont, C. et al. J. Infect. Dis. 2000, 181: 1996-2002. Nguyen VH et al., Cancer Res. 2010, 1; 70(1): 18-23, Kim K et al., PLoS One. 2013, 8(3): e60511).

[0008] However, anticancer treatment using probiotic strains has been concerned about the occurrence of side effects due to the fact that the strains are also retained in normal tissues other than tumor tissues. Specifically, recombinant Salmonella used to treat tumors produces strong anticancer substances, which can cause tissue damage due to the retention of Salmonella in normal tissues. In addition, when the remaining Salmonella that is not eliminated causes chronic infection to occur after tumor cells die, carriers can cause infection in others. In addition, pathogenic attenuated Salmonella can bring fatal sepsis to patients or the elderly with low immunity, and the antibodies produced during chronic infection can cause autoimmune diseases such as arthritis, ophthalmia, urethritis, etc. Therefore, in the development of a technology for treating cancer using Salmonella, there is a need to develop a Salmonella strain that can be rapidly eliminated from normal organs after Salmonella infection, effectively control the activity of Salmonella in normal tissues other than tumor tissues by increasing the specificity of Salmonella to tumor tissues and the proliferation of Salmonella in tumor tissues, and minimize excessive immune responses in normal organs and side effects caused thereby. It has been reported that mutant strains lacking pathogenic genes of Salmonella regulate the virulence and activity of Salmonella. Inactivation of the pathogenic gene cluster of Salmonella, Salmonella pathogenicity island-1 (SPI-1) or Salmonella pathogenicity island-2 (SPI-2), showed the effect of attenuating the pathogenicity of Salmonella gallinarum (Korean Patent No. 1015008050000), and the survival ability of the mutant strain in the spleen was rapidly reduced after additional deletion of a single gene belonging to the pathogenic gene cluster in attenuated Salmonella (Korean Patent No. 1017735800000).

[0009] However, the deletion of the SPI-1 or SPI-2 gene clusters in Salmonella chickens, resulting in non-pathogenicity due to reduced penetration efficiency in avian epithelial cells, cannot be considered a rapid modulation of Salmonella activity in all normal tissues outside of immune organs, as the activity of residual Salmonella in normal tissues cannot be confirmed. The reduced activity of Salmonella in the spleen, on the other hand, is due to the deletion of a single gene belonging to a pathogenic gene cluster in attenuated Salmonella. This reduced activity in the spleen is thought to be due to increased susceptibility of the spleen (an immune organ composed of lymphocytes and various immune cells) to Salmonella.

[0010] Therefore, the inventors have strived to develop a Salmonella strain with effectively controlled activity in normal tissues other than tumor tissue and excellent anticancer activity. The results showed that when the pathogenic gene clusters of Salmonella were additionally deleted from previously known attenuated Salmonella strains, the resulting strain significantly inhibited tumor growth while exhibiting reduced activity in normal organs. This indicates that the strain of the present invention can be effectively used as an active ingredient in anticancer compositions, thus completing the present invention.

[0011] public

[0012] Technical issues

[0013] The purpose of this invention is to provide a gene-deleted Salmonella mutant strain and a method for generating the mutant strain.

[0014] Another object of the present invention is to provide a composition containing the Salmonella mutant strain provided by the present invention for the prevention, improvement or treatment of cancer.

[0015] However, the objectives of this invention are not limited to those described above. Other objectives not mentioned in this invention will be clearly understood by those skilled in the art through the following description.

[0016] Technical solution

[0017] In the following description, various embodiments described herein will be described with reference to the accompanying drawings. Numerous specific details, such as specific configurations, compositions, and processes, are set forth in the description below to provide a comprehensive understanding of the invention. However, some embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparation techniques have not been described in detail to avoid unnecessarily obscuring the invention. The phrase "one embodiment" or "one implementation" as referenced throughout this specification means that a specific feature, configuration, combination, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the phrase "in one embodiment" or "one implementation" in different places in this specification does not necessarily refer to the same embodiment of the invention. Furthermore, features, configurations, combinations, or characteristics may be combined in any suitable manner in one or more embodiments.

[0018] Unless otherwise stated in the specification, all scientific and technical terms used in the specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] According to an embodiment of the present invention, the present invention relates to a Salmonella mutant strain wherein Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2) are deleted.

[0020] In this invention, Salmonella pathogenicity island-1 (SPI-1) is a specific region in the Salmonella genome that contains a type III secretion system, which is essential for early invasion of intestinal epithelial cells by Salmonella infection, as well as a gene cluster encoding virulence proteins (Kimbrough TG et al., Microbes Infect, (2002); 4(1):75-82).

[0021] In this invention, Salmonella pathogenicity island-1 (SPI-1) may be composed of the nucleotide sequence described in SEQ ID NO:1.

[0022] In this invention, Salmonella pathogenicity island-2 (SPI-2) is a specific genomic region containing: effector proteins involved in the mechanism by which Salmonella survives in the phagocytosis of macrophages after translocation through epithelial cells in intestinal immune organs or immune organs (e.g., spleen and liver) without being lysed in the phagosomes; and the type III secretion system that secretes these proteins (Waterman SR et al., Cell Microbiol, (2003); 5(8):501-511, Abrahams GL, Cell Microbiol, (2006); 8(5):728-737). The type III secretion system (TTSS) is a pathogenic translocation mechanism formed in Gram-negative bacteria. It is a multi-protein complex that is a syringe-like structure that directly injects pathogenic active proteins into the cytoplasm through the host cell membrane (Mota LJ et al., Ann Med. (2005); 37(4):234-249). The translocation of pathogenic substances via the type III secretory system is known to be crucial in the Salmonella infection pathway (Schlumberger MC et al., Curr Opin Microbiol, (2006); 9(1):46-54). Wild-type Salmonella uses the TTSS protein to adhere to and invade the host's intestinal epithelial cells, surviving even during macrophage phagocytosis and, in some cases, spreading via the bloodstream, causing systemic infection. Therefore, Salmonella infection progresses when TTSS protein function is normal.

[0023] In this invention, Salmonella pathogenicity island-2 (SPI-2) may be composed of the nucleotide sequence shown in SEQ ID NO:2.

[0024] In this invention, Salmonella mutant strains may be strains that further incorporate genes encoding anticancer proteins.

[0025] As used in this invention, the term "anticancer protein" refers to a peptide that can directly or indirectly induce cancer cell death. The anticancer protein may be, for example, at least one selected from the group consisting of: toxin proteins, specific antibodies or antibody fragments against cancer antigens, tumor suppressor proteins, angiogenesis inhibitors, cancer antigens, prodrug-converting enzymes, and pro-apoptotic proteins, but is not limited thereto.

[0026] As used in this invention, the term "toxin protein" refers to a protein that can directly or indirectly induce cancer cell death. For example, a toxin protein may be at least one selected from the group consisting of: ricin, saponins, white tree toxin, momordicin, debouganin, diphtheria toxin, pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA). Preferably, the toxin protein may be cytolysin A, but is not limited thereto.

[0027] In this invention, the introduction of the cytolysin A gene can be carried out through a recombinant vector, but is not limited thereto.

[0028] In this invention, the recombinant vector can be prepared using the forward primer represented by SEQ ID NO: 13 and the reverse primer represented by SEQ ID NO: 14.

[0029] In this invention, the cytolysin A (ClyA) gene is a gene encoding a 34-kDa protein that lyses cells by forming pores in the cell membrane and is expressed in *Escherichia coli*, *Salmonella*, etc. The sequence of cytolysin A is described in Waisn, Lindmark B, Soderblom T, et al., vesicle-mediated pore-forming oligomer export and assembly of *Enterobacterial* ClyA cytotoxin, Cell 2003;115:25-35, and is not limited thereto, as are those derived from various prokaryotic cells and their functional variants, which may be referred to herein as cytolysin A. In one embodiment, the protein sequence of cytolysin A (ClyA) is available from accession number ABI83833.1, and its gene sequence is available from accession number DQ910780.1. The cytolysin A gene may consist of the nucleotide sequence shown in SEQ ID NO: 15.

[0030] The term "tumor suppressor protein" as used in this invention refers to a gene that maintains its function in normal cells, but when it loses its function, allows normal cells to divide arbitrarily and grow into cancer cells. Examples of tumor suppressor proteins include, but are not limited to, retinoblastoma (RB) protein, p53 protein, adenomatous polyposis (APC) protein, homologous phosphatase and tensin (PTEN), cyclin-dependent kinase inhibitor 2A (CDKN2A) protein, etc.

[0031] In this invention, the specific antibodies and antibody fragments targeting cancer antigens are antibodies capable of specifically binding to antigens that are proteins with specific expression levels on the surface or cytoplasm of cancer cells. For example, the antibody or its fragment may be a specific antibody against HER2, which is specifically highly expressed in breast or gastric cancer cells, but is not limited thereto.

[0032] As used in this invention, "antibody" refers to a protein molecule capable of specifically binding to an antigenic site on a protein or peptide molecule. There are no particular limitations on the type of antibody, and examples include polyclonal antibodies, monoclonal antibodies, or antibody fragments with antigen-binding properties, and include all types of immunoglobulin antibodies. Furthermore, examples of antibodies include specific antibodies such as humanized antibodies. Examples of antibodies include not only whole antibodies having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. The "functional fragment of an antibody molecule" refers to a fragment with at least antigen-binding function, and examples include, but are not limited to, Fab, F(ab′), F(ab′)2, Fv, etc.

[0033] The antibody of the present invention can be prepared by cloning the gene encoding the cancer antigen of the present invention into an expression vector using conventional methods, and then obtaining the protein encoded by the gene using conventional methods.

[0034] As used in this invention, the term "angiogenesis inhibitor" refers to a protein or compound that can directly or indirectly induce cancer cell death by inhibiting the formation of new blood vessels around cancer cells. Preferably, examples of angiogenesis inhibitors include, but are not limited to, angiostatin, endostatin, thromboreactive protein, and protease inhibitory protein.

[0035] The term "cancer antigen" as used in this invention refers to a protein antigen expressed in cancer cells but rarely expressed in normal cells, thereby inducing an anti-tumor immune response and subsequently inducing the direct or indirect death of cancer cells. Preferably, the cancer antigen of this invention may be α-fetoprotein (AFP), vascular endothelial growth factor receptor 2 (VEGFR2), Survivin, asparagine endopeptidase (Legumain), prostate cancer-specific antigen (PCSA), etc., but is not limited thereto.

[0036] The term "prodrug-converting enzyme" as used in this invention refers to a protein that has the function of converting an inactive drug into an active drug through enzymatic metabolism. When such a prodrug-converting enzyme is used, the inactive drug can be metabolized and converted into an active drug that can directly or indirectly induce cancer cell death. Therefore, prodrug-converting enzymes may be very useful for the prevention or treatment of cancer. Preferred examples of prodrug-converting enzymes of this invention include, but are not limited to, thymidine kinase, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, carboxypeptidase G2, chromate reductase YieF, herpes simplex virus type I thymidine kinase / ganciclovir (HSV1-TK / GCV), β-glucuronidase, etc.

[0037] As used in this invention, the term "pro-apoptotic protein" refers to a protein that induces the direct or indirect death of cancer cells by depriving them of factors (proteins, nutrients, oligonucleotides, etc.) essential for their growth or maintenance. Preferred examples of pro-apoptotic proteins of this invention include, but are not limited to, L-ASNase and RNA-binding motif 5 (RBM5).

[0038] In this invention, the Salmonella mutant strain may also contain a reporter gene, and the reporter gene is capable of imaging the Salmonella mutant strain.

[0039] Reporter genes encode proteins that can be imaged in vivo or in vitro, and examples include bioluminescent genes, chemiluminescent genes, and fluorescent genes from various sources.

[0040] In this invention, the luminescent gene can be a luciferase gene derived from prokaryotic or eukaryotic cells. A luciferase gene derived from prokaryotic cells can be a bacterial luciferase gene (lux). A luciferase gene derived from eukaryotic cells can be a firefly-derived luminescent gene, but is not limited thereto. This gene can exist in plasmid form or be inserted into the genome of Salmonella.

[0041] In this invention, the luminescent gene can exist in the form of a plasmid or be inserted into the genome of Salmonella.

[0042] In this invention, the Salmonella mutant strain can be derived from at least one selected from the group consisting of: *Salmonella typhimurium*, *Salmonella choleraesuis*, *Salmonella enteritidis*, *Salmonella infantis*, *Salmonella paratyphi*, and *Salmonella typhi*. The listed Salmonella strains are Gram-negative facultative anaerobic bacteria. They differ from *Escherichia coli* in that they lack lactose degradation capabilities, do not form indole, and do not produce hydrogen sulfide. The Salmonella strains can be active.

[0043] Salmonella typhimurium is a strain of Salmonella that causes typhoid fever. It is a flagellated rod-shaped spore-forming bacillus that is Gram-negative. Salmonella typhimurium is relatively heat-sensitive, dying within 20 minutes at 60°C. Livestock, wild animals, humans, milk, and eggs are likely to be primarily contaminated with Salmonella typhimurium. Salads and similar foods can easily become secondary infections due to contaminated meat, potentially leading to salmonellosis, a form of food poisoning.

[0044] Salmonella cholerae is a well-known Salmonella bacterium that causes swine fever and infects humans and animals. It is a leading cause of acute septicemia caused by Salmonella. It is a Gram-negative facultative anaerobic bacillus with flagella, enabling motility. It differs from Escherichia coli in that it lacks lactose-degrading ability, does not form indole, and does not produce hydrogen sulfide. Salmonella cholerae grows optimally at temperatures between 35 and 37°C, can multiply at temperatures between 10 and 43°C, and is killed by heating at 60°C for 20 minutes. The optimal pH for Salmonella cholerae is 7.2 to 7.4, and its size is 0.5 to 0.8 x 3 to 4 μm.

[0045] Salmonella enteritidis is a type of Salmonella bacterium that causes food poisoning. Also known as Bacillus enteritidis, it is a representative Salmonella bacterium that can infect various animals and exhibits high adaptability to its host. It is a Gram-negative facultative anaerobic bacillus with flagella, enabling it to move. It differs from Escherichia coli in that it lacks the ability to degrade lactose, does not form indole, and does not produce hydrogen sulfide. Salmonella enteritidis grows optimally at temperatures between 35 and 37°C, can multiply at temperatures between 10 and 43°C, and is killed by heating at 60°C for 20 minutes. The optimal pH for Salmonella enteritidis is 7.2 to 7.4, and its size is 0.5 to 0.8 x 3 to 4 μm.

[0046] Salmonella infantis is a bacterium that infects eggs or poultry, while Salmonella paratyphi and Salmonella typhi are the bacteria that cause typhoid fever.

[0047] In this invention, the Salmonella mutant strain can be a mutant strain lacking the ability to synthesize guanosine polyphosphate. The guanosine polyphosphate can be guanosine-5-bisphosphate-3-bisphosphate (ppGpp).

[0048] In this invention, the Salmonella mutant strain can be a strain in which at least one of the Salmonella-relA gene and the Salmonella-spoT gene encoding ppGpp synthase is inactivated.

[0049] In this invention, Salmonella strains with the ppGpp synthesis gene deleted exhibit tumor-suppressive activity, but can also show significant viability and cause Salmonella infection even in normal organs and tissues. Furthermore, strains with only the Salmonella pathogenicity gene deleted retain their pathogenicity in normal organs, thus their potential use as therapeutic strains is low. However, the Salmonella gene of this invention, obtained by additionally deleting the SPI-1 and SPI-2 genes from the Salmonella gene to inactivate the ppGpp synthesis gene, exhibits significantly low viability in normal organs, thus not causing Salmonella infection, and has the advantage of showing significantly better anti-cancer effects than Salmonella strains with the ppGpp synthesis gene deleted.

[0050] In this invention, the Salmonella mutant strain can be derived from SHJ2037 (accession number KCTC 10787BP).

[0051] In this invention, the term "derived" refers to an existing plasmid or strain that has not undergone a genetic recombination process to produce a recombinant plasmid or recombinant strain.

[0052] To produce the gene-deleted and inactivated Salmonella mutant strains of the present invention, a recombinant vector can be used in Salmonella host cells. The recombinant vector can be a plasmid, but is not limited thereto. The plasmid can be introduced into the Salmonella strain by electroporation, but is not limited thereto. Electroporation is a method of applying an electric shock to protoplasts to introduce DNA into the cell membrane. When a high-voltage electrical stimulus is applied to the cell at regular intervals, a small pore is opened in the cell membrane, through which DNA is inserted into the cell by electrophoresis.

[0053] The Salmonella mutant strain of the present invention can be used for the prevention or treatment of cancer.

[0054] In this invention, "cancer" is a disease characterized by the rapid and uncontrolled growth of mutated cells, and may be at least one selected from the group consisting of: melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, leukemia, thyroid cancer, endocrine gland cancer, oral cancer, liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma. Preferably, the cancer may be at least one selected from the group consisting of: liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone / soft tissue sarcoma, and skin cancer. More preferably, the cancer may be colon cancer, but is not limited thereto.

[0055] The Salmonella mutant strains of this invention may exhibit lower viability in normal tissues than in tumor tissues. Here, normal tissues may be selected from, but are not limited to, tissues belonging to the group of organs including the lungs, liver, and spleen.

[0056] Because the Salmonella mutant strain of the present invention has significantly lower viability in normal tissues, it does not cause Salmonella infection and has the advantage of exhibiting significantly better anti-cancer effects than conventional inventions.

[0057] Furthermore, it is understood that if the Salmonella mutant strain of the present invention is attenuated by two or more different mutations in the genome, it is safe because the possibility of the Salmonella mutant strain reverting to the parent strain is very low.

[0058] According to another embodiment of the present invention, a method for producing a Salmonella mutant strain is provided, the method comprising the step of deleting the Salmonella pathogenic island-1 (SPI-1) and Salmonella pathogenic island-2 (SPI-2) genes from a Salmonella strain to obtain a transformed strain.

[0059] The Salmonella pathogenicity island-1 (SPI-1) of this invention can be composed of the nucleotide sequence shown in SEQ ID NO: 1.

[0060] The Salmonella pathogenicity island-2 (SPI-2) in this invention may be composed of the nucleotide sequence shown in SEQ ID NO: 2.

[0061] In this invention, the deletion of the Salmonella pathogenic island-1 (SPI-1) and Salmonella pathogenic island-2 (SPI-2) genes can be performed using a recombinant vector, but is not limited thereto.

[0062] In this invention, the recombinant vector can be prepared using the forward primer shown in SEQ ID NO: 7 and the reverse primer shown in SEQ ID NO: 8.

[0063] The recombinant vector can be derived from pKD13, pCP20 or pJL39 plasmids, but is not limited to these.

[0064] The pKD13 plasmid is 3434 bp in length and contains β-lactamase, rrnB transcription terminator, primer site 1, native FRT site, universal primer site kt, Tn5 neomycin phosphotransferase, universal primer site k2, universal primer site k1, 35-nt distal to the native FRT site, primer site 4, λ terminator, and R6Kγ replication origin gene.

[0065] The pCP20 plasmid is 9497 bp in length and contains the EcoRI, cat, Pstl, HindIII Ci857, flp, bamHi, β-lactamase, mobA, mob2, and repA101ts genes.

[0066] The preparation method according to the present invention may further include the step of introducing a gene encoding an anticancer protein.

[0067] In this invention, the anticancer protein may be at least one selected from the group consisting of toxin proteins, cancer antigen-specific antibodies, antibody fragments, tumor suppressor proteins, angiogenesis inhibitors, cancer antigens, prodrug converting enzymes, and proapoptotic proteins, but is not limited thereto.

[0068] In this invention, the toxin protein may be at least one selected from the group consisting of: ricin, saponin, white tree toxin, momordicin, debouganin, diphtheria toxin, pseudomonadine, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA). Preferably, the toxin protein may be cytolysin A, but is not limited thereto.

[0069] In this invention, the introduction of the cytolysin A gene can be performed using a recombinant vector, but is not limited thereto. The vector used to introduce the cytolysin A gene can be the same as or different from the vector used to delete the SP-1 and SP-2 genes.

[0070] In this invention, the recombinant vector can be prepared using the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 14.

[0071] In this invention, the introduction of plasmids of recombinant vectors can be performed by electroporation, but is not limited thereto.

[0072] In this invention, the step of introducing the cytolysin A (ClyA) gene can be performed before or after the deletion of the Salmonella Pathogenic Island-1 (SPI-1) and Salmonella Pathogenic Island-2 (SPI-2) genes, and there is no particular restriction on the order of the steps.

[0073] In this invention, the Salmonella mutant strain may be derived from at least one of the following groups: Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, and Salmonella typhi.

[0074] In this invention, the Salmonella mutant strain can be a mutant strain that lacks the ability to synthesize guanosine polyphosphate.

[0075] In this invention, the Salmonella strain may be a strain in which at least one of the Salmonella-relA gene and Salmonella-spoT gene encoding ppGpp synthase is inactivated.

[0076] The preparation method of the present invention may further include the step of culturing the above-mentioned transformed strain.

[0077] In this invention, the culturing step can be carried out using lysogenic broth (LB) containing antibiotics, and the lysogenic broth (LB) was developed by Giuseppe Bertani to optimize the growth and plaque formation of Shigella strains. Lysogenic broth (LB) generally contains peptides, casein peptone, vitamins (including B vitamins), trace elements (nitrogen, sulfur, magnesium), and minerals, and its osmotic pressure is controlled by sodium chloride.

[0078] The preparation method according to the present invention may further include the steps of culturing the above-mentioned transformed strain in an antibiotic culture medium and selecting mutant strains.

[0079] In this invention, the antibiotic can be kanamycin or tetracycline, but is not limited to these.

[0080] Detailed information regarding the Salmonella mutant strains, anticancer proteins, Salmonella strains, and recombinant vectors of the present invention is the same as that of the compositions described above, and therefore will be omitted to avoid making this specification overly complicated.

[0081] According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, wherein the present invention contains a Salmonella mutant strain as an active ingredient.

[0082] In this invention, "cancer" is a disease characterized by the rapid and uncontrolled growth of mutated cells, and may be at least one selected from the group consisting of: melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, leukemia, thyroid cancer, endocrine gland cancer, oral cancer, liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone / soft tissue sarcoma, skin cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma. Preferably, the cancer may be at least one selected from the group consisting of: liver cancer, biliary tract cancer, colorectal cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone / soft tissue sarcoma, and skin cancer. More preferably, the cancer may be colon cancer, but is not limited thereto.

[0083] The term "prevention" as used in this invention may include, but is not limited to, any effect of blocking or suppressing or delaying symptoms caused by cancer by using the compositions of this invention.

[0084] The terms “improvement” or “treatment” used in this invention may include, but are not limited to, any effect that beneficially alters symptoms caused by cancer or benefits an individual with such symptoms through the use of the active ingredients of this invention.

[0085] The pharmaceutical compositions of the present invention may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical compositions may be intended for administration to humans.

[0086] For their intended use, the pharmaceutical compositions of the present invention can be formulated according to their respective conventional methods, but are not limited to, oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., and topical preparations, suppositories, and sterile injections. The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers for oral administration include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings, etc. Pharmaceutically acceptable carriers for injection include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. Pharmaceutically acceptable carriers for topical administration include alkalis, excipients, lubricants, preservatives, etc. The dosage forms of the pharmaceutical compositions of the present invention can be prepared in various ways by mixing pharmaceutically acceptable carriers as described above. For example, for oral administration, the pharmaceutical compositions can be prepared in the form of tablets, granules, capsules, elixirs, suspensions, syrups, or wafers; for injection, the pharmaceutical compositions can be presented in single-dose ampoules or multi-dose containers. Furthermore, the pharmaceutical composition can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0087] Examples of suitable carriers, excipients, and diluents for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl methylbenzoate, propylparaben, talc, magnesium stearate, and mineral oil. Furthermore, the pharmaceutical composition may also contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0088] The routes of administration for the pharmaceutical compositions of the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, local, sublingual, and rectal routes. Oral or parenteral administration is preferred.

[0089] In this invention, "parenteral" includes subcutaneous, transdermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can also be formulated into suppositories for rectal administration.

[0090] The pharmaceutical compositions of the present invention can vary depending on various factors, including the activity of the specific compound used, the patient's age, weight, general health condition, sex, diet, time of administration, route of administration, excretion rate, drug content, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition can be appropriately selected by those skilled in the art based on the patient's condition, weight, disease severity, form of the drug, and route and duration of administration, and can be from 0.0001 to 50 mg / kg / day or 0.001 to 50 mg / kg / day. The pharmaceutical composition can be administered once or several times daily. The dosage is not intended to limit the scope of the invention in any way. The pharmaceutical compositions according to this disclosure can be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, liquids, or suspensions.

[0091] In another embodiment of the invention, a composition for diagnosing cancer is provided, comprising a Salmonella mutant strain as an active ingredient.

[0092] The diagnostic composition of the present invention contains the strain of the present invention as an active ingredient.

[0093] After the mutant strain of this invention targets cancer cells in an individual, it is able to express a reporter protein for real-time imaging within the strain. Therefore, this strain can diagnose cancer in real time without noise signals from normal tissue.

[0094] As used in this invention, the term "reporter protein" refers to a protein whose function enables visual diagnosis of cancer. For example, a reporter protein may be, but is not limited to, at least one selected from the group consisting of fluorescent proteins, luciferases, and proteins used in nuclear medicine or MRI imaging.

[0095] As used in this invention, the term "fluorescent protein" refers to a protein that emits fluorescence so that it can be visually diagnosed as cancer. For example, the fluorescent protein may be at least one selected from the group consisting of: green fluorescent protein (GFP), modified green fluorescent protein (MGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), and enhanced yellow fluorescent protein (EYFP), but is not limited thereto.

[0096] In this invention, the protein used for nuclear medicine or MRI imaging may be, for example, at least one selected from the group consisting of: herpes simplex virus thymidine kinase, dopamine receptor, somatostatin receptor, sodium iodide transporter, iron receptor, transferrin receptor, ferritin and iron transporter (magA), but is not limited thereto.

[0097] The term "diagnosis" as used in this invention refers to any action that detects cancerous tissue in vivo, including, when the strains of this invention are targeted to cancer, real-time monitoring of the presence of cancer by expressing reporter proteins from DNA constructs introduced into the strains.

[0098] In the diagnostic compositions of the present invention, the detailed information regarding mutant strains, vectors, transformations, cancers, etc., is the same as described above, and therefore, repeated descriptions will be omitted to avoid making this specification overly complicated.

[0099] In another embodiment of the invention, a method for providing information for diagnosing cancer is provided, comprising the step of treating a biological sample with a Salmonella mutant strain.

[0100] The method of the present invention includes the step of treating biological samples isolated from subjects of interest with the mutant strain of the present invention.

[0101] The method for providing information for diagnosing cancer according to the present invention may further include the step of diagnosing cancer when a reporter protein is expressed from a strain.

[0102] As used in this invention, the term "biological sample" refers to any material, tissue, or cell obtained from or derived from a subject. Examples of biological samples include, but are not limited to, tissues, cells, or cell extracts.

[0103] In the method for providing diagnostic information according to the present invention, the detailed information regarding mutant strains, anticancer proteins, reporter proteins, vectors, strains, transformation, cancer, diagnosis, etc., is the same as the detailed information regarding reporter proteins, vectors, strains, and pharmaceutical compositions for the prevention or treatment of cancer described above, and therefore, repeated descriptions will be omitted to avoid making this specification overly complicated.

[0104] Another embodiment of the invention relates to a method for preventing or treating cancer, the method comprising the step of administering to a subject an effective amount of a Salmonella mutant strain provided by the invention.

[0105] As used in this invention, the term "subject" refers to an individual who needs cancer prevention or treatment, and may include both mammals and non-mammals. Examples of mammals include, but are not limited to, humans; non-human primates such as chimpanzees, or other ape or monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, or cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals in this invention include, but are not limited to, birds or fish.

[0106] As used herein, the term "administration" refers to the process of delivering the active ingredient of the present invention to a subject by any suitable method. There are no particular limitations on the dosage form of the Salmonella mutant strains administered as described above, and the mutant strains can be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, or as a solid formulation intended to be immediately converted to a liquid form before use for oral or parenteral administration. For example, the pharmaceutical compositions of the present invention can be formulated into forms for administration, but not limited to, oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical formulations, suppositories, and sterile injectable solutions.

[0107] Furthermore, in this invention, pharmaceutically acceptable carriers can be additionally administered together with the Salmonella mutant strain of this invention. Examples of pharmaceutically acceptable carriers suitable for oral administration include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc. Examples of pharmaceutically acceptable carriers suitable for injection include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. Examples of pharmaceutically acceptable carriers suitable for topical administration include alkalis, excipients, lubricants, preservatives, etc. The dosage form of the Salmonella mutant strain of this invention can be prepared in various ways by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition can be prepared in the form of tablets, capsules, elixirs, suspensions, syrups, wafers; for injection, the pharmaceutical composition can be prepared in unit dose form or prepared into multi-dose containers. Furthermore, the pharmaceutical composition can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0108] Examples of suitable carriers, excipients, and diluents for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl methylbenzoate, propylparaben, talc, magnesium stearate, and mineral oil. Furthermore, the pharmaceutical composition may also contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0109] The Salmonella mutant strains of the present invention can be administered via routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, local, sublingual, and rectal routes. Oral or parenteral administration is preferred.

[0110] In this invention, "parenteral" includes subcutaneous, transdermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can also be formulated into suppositories for rectal administration.

[0111] In this invention, "effective amount" refers to a sufficient quantity of reagent to provide a desired biological outcome. This outcome can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. For example, an effective amount for therapeutic use is the amount of the Salmonella mutant strain of this disclosure to provide clinically significant disease remission. In any individual case, an appropriate "effective" amount can be determined by those skilled in the art using routine experiments. Therefore, the expression "effective amount" generally refers to the amount of an active substance that has a therapeutic effect. In the context of this invention, the active substance is a reagent used for the prevention, improvement, or treatment of cancer.

[0112] The Salmonella mutant strains of the present invention can vary depending on various factors, including the activity of the specific compound used, the patient's age, weight, general health condition, sex, diet, time of administration, route of administration, excretion rate, drug content, and the severity of the specific disease to be prevented or treated. The dosage of the Salmonella mutant strain can be appropriately selected by those skilled in the art based on the patient's condition, weight, disease severity, drug form, and route and time of administration, and can be 0.0001 to 100 mg / kg / day or 0.001 to 100 mg / kg / day. The Salmonella mutant strain can be administered once or several times daily. The stated dosage is not intended to limit the scope of the invention in any way. The Salmonella mutant strains of the present invention can be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

[0113] In this invention, the detailed description of cancer is the same as described above, and therefore its detailed description will be omitted to avoid making this specification too complicated.

[0114] The Salmonella mutant strains of the present invention can be used alone or in combination with methods such as surgery, radiotherapy, hormone therapy, chemotherapy and biological response modifiers.

[0115] Beneficial effects

[0116] The gene-deleted Salmonella mutant strain of the present invention exhibits very low viability in normal tissues, while surviving in cancerous tissues and effectively inducing cancer cell death, thus demonstrating excellent anti-cancer effects. Unlike Salmonella strains, it has the advantage of not causing Salmonella infection.

[0117] Brief description of the attached figures

[0118] Figure 1This is a schematic diagram of the method for constructing the pJH18 plasmid.

[0119] Figure 2 This is a graph showing the growth rate of a recombinant strain containing the pJH18 plasmid.

[0120] Figure 3 It is a graph showing the growth rate of the strain.

[0121] Figure 4 The results of analyzing the expression of reporter proteins for each Salmonella strain are shown.

[0122] Figure 5 The images show the results of luciferase activity measured in each cultured strain by adding coelenterazine.

[0123] Figure 6 It is a graph showing the luciferase activity in each cultured strain.

[0124] Figure 7 The results show the extent of hemolytic activity of the strain on blood agar.

[0125] Figure 8 A graph showing the measurement results of ATP in a culture according to an embodiment of the present invention is displayed.

[0126] Figure 9 The results of the analysis of the distribution of Salmonella in mouse livers are shown.

[0127] Figure 10 The results of the analysis of Salmonella distribution in mouse tumors are shown.

[0128] Figure 11 The process of extracting spleens from tumor animal models treated with each strain is shown.

[0129] Figure 12 The results show the spleen size measurements of tumor-bearing animals treated with each strain.

[0130] Figure 13 A graph showing the results of measuring spleen weight in tumor animals treated with each strain.

[0131] Figure 14 This is a tumor volume map measured after an animal model of tumors injected with CT26 was used, with CNC18 as an example of the present invention and PBS and SHJ2037 as controls.

[0132] Figure 15 This is a graph showing the viability results of each strain after inoculation in a tumor animal model injected with CT26.

[0133] Figure 16This is a graph showing the tumor volume measured after an animal model of tumors was inoculated with MC38, using CNC18 as an example of the present invention and PBS and SHJ2037 as controls.

[0134] Figure 17 This is a graph showing the viability results of each strain after inoculation in a tumor animal model injected with MC38.

[0135] Figure 18 The graph shows the results of measuring and comparing the proportion of white blood cells in tumors after each strain was injected into an animal tumor model.

[0136] Figure 19 The graph shows the results of measuring and comparing the proportion of neutrophils in tumors after each strain was injected into an animal tumor model.

[0137] Figure 20 This is a graph showing the results of measuring and comparing the proportion of natural killer cells in tumors after each strain was injected into an animal tumor model.

[0138] Figure 21 This is a graph showing the results of measuring and comparing the proportion of CD8+ T cells in tumors after each strain was injected into an animal tumor model.

[0139] Figure 22 This is a graph showing the results of measuring and comparing the proportion of dendritic cells in lymph nodes after each strain was injected into a tumor animal model.

[0140] Figure 23 This is a graph showing the results of measuring and comparing the proportion of M2 macrophages in tumors after each strain was injected into an animal tumor model.

[0141] Figure 24 This is a graph showing the results of measuring and comparing the proportion of regulatory T (Treg) cells in tumors after each strain was injected into an animal tumor model.

[0142] Figure 25 Images of tumors and organs extracted on day 1 after various strains of SHJ2037lux and CNC18lux were injected into tumor animal models, obtained using an in vivo imaging system (IVIS).

[0143] Figure 26 Images of tumors and organs extracted on day 3 after various strains of SHJ2037lux and CNC18lux were injected into tumor animal models, obtained using an in vivo imaging system (IVIS).

[0144] Figure 27Images of tumors and organs extracted on day 5 after various strains of SHJ2037lux and CNC18lux were injected into tumor animal models, obtained using an in vivo imaging system (IVIS).

[0145] Figure 28 Images obtained using an in vivo imaging system (IVIS) are shown after each strain of SHJ2037lux and CNC18lux was injected into an animal model of multiple myeloma.

[0146] Figure 29 Images obtained using an in vivo imaging system (IVIS) are shown after each strain of SHJ2037lux and CNC18lux was injected into an animal model of multiple myeloma.

[0147] Best way

[0148] According to an embodiment of the present invention, the present invention relates to a Salmonella mutant strain wherein Salmonella pathogenic island-1 (SPI-1) and Salmonella pathogenic island-2 (SPI-2) are deleted.

[0149] Invention Embodiments

[0150] The invention will be described in more detail below with reference to embodiments. These embodiments are only for explaining the invention in more detail, and it will be apparent to those skilled in the art that the scope of protection of the invention is not limited to these embodiments.

[0151] [Preparation Example 1] Construction of Attenuated Salmonella

[0152] The attenuated Salmonella strain of the present invention was constructed using SHJ2037 strain as a template, and pathogenicity-related genes or gene clusters were deleted by homologous recombination using lambda(λ) phage.

[0153] Specifically, the pKD13 plasmid was used to amplify DNA fragments with Kan boxes at the open reading frame positions of each gene by PCR. The PCR primer sequences used to knock out a total of three genes (clusters) are shown in Table 1 below. The PCR product pKD46 was introduced into a suspension of Salmonella Typhimurium SMR2130 (SHJ2037, DrelA, DspoT) containing cells by electroporation, and colonies were selected by culturing in kanamycin medium. Then, the pCP20 plasmid (Datsenko KA et al., Proc Natl Acad Sci US A.2000, 6; 97(12): 6640-5) was introduced into the transformed strain by electroporation, and the Kan boxes were knocked out by FLP recombinase to construct the strain.

[0154] [Table 1]

[0155]

[0156] [Preparation Example 2] Construction of recombinant Salmonella for gene expression

[0157] Figure 1 A schematic diagram of the method for constructing the pJH18 plasmid is shown. Using the pJL39 plasmid (Mol Ther., 21(11), p. 1985-1995, (2013)) as the template strand, the tetR gene was amplified using a constructed forward primer containing the restriction endonuclease EcoRI site (5'-CGGAATTCACCATGTCTAGATTAGAAAAGTAAAGTGATTAACAG-3'; SEQ ID NO: 9) and a constructed reverse primer containing the restriction endonuclease PvuII-XbaI site (5'-GCTCTAGACAGCTGTTAAGACCCACTTTCACATTTAAGTTTTTTTT-3'; SEQ ID NO: 10). Subsequently, the amplification products were digested with restriction endonucleases EcoRI and XbaI and purified to obtain the tetR gene amplification product, which was then introduced into pBAD24 (catalog number...). 87399 TM The pBAD-TetR plasmid was constructed by incorporating the plasmid from ATCC (United States).

[0158] Subsequently, different promoter regions containing multiple cloning sites were introduced into the pBAD-TetR plasmid using the PvuII and HindIII fragments of the pJL39 plasmid, thus constructing the pTetR-BAD plasmid. The araC and araBAD promoters were then removed from the pTetR-BAD plasmid using NheI and Pcil restriction endonucleases, thus constructing the pTetII plasmid.

[0159] In addition, using pSF-OXB1 (Oxford Genetics, UK) as a template, and with forward primer (5'-CTACTCCGTCAAGCCGTCAAGCTGTGTGTGACCGCTTGCT-3'; SEQ ID NO: 11) and reverse primer (5'-TGAATTCCTCCTGCTAGCTAGTTGGTAACGAATCAGACGCCGGGTAATACCGGATAG-3'; SEQ ID NO: 12) to amplify the constitutive promoter OXB1, it was introduced into the pTetII plasmid using the Gibson assembly method, thereby constructing the pJH18 plasmid containing the OXB1, tetA and tetR promoters.

[0160] Using pJH18 plasmid as a template, the genes encoding Rluc8 and cytolysin A (ClyA) were introduced into... Figure 1 Downstream of the promoter shown in the diagram. Thus, the pJH18-CR plasmid was constructed. Here, the cytolysin A (ClyA) gene was introduced using a forward primer (5′-AGTCCATGGTTATGACCGGAATTTGC-3′(SEQ ID NO: 13)) and a reverse primer (5′-GATGTTTAAACTCAGACGTCAGGAACCTC-3′(SEQ ID NO: 14)).

[0161] [Preparation Example 3] Construction of a transformed Salmonella mutant strain

[0162] The plasmid constructed in Preparation Example 2 was transformed into the Salmonella strain constructed in Preparation Example 1 via electroporation, and each transformed strain was then cultured overnight in lysogenic broth (LB) solid medium containing 100 μg / ml ampicillin. Subsequently, the resulting bacterial population, cultured in LB liquid medium containing ampicillin, was used for experiments. Table 2 below summarizes the transforming components in the Salmonella mutant strains constructed as described above.

[0163] [Table 2]

[0164] Mutant strain Deletion gene Insertion gene Comparative Example 1 SHJ2037 relA, spoT ClyA, Rluc8 Comparative Example 2 CNC16 relA, spoT, hilD ClyA, Rluc8 Comparative Example 3 CNC17 relA, spoT, SPI 1 ClyA, Rluc8 Preparation Example CNC18 relA, spoT, SPI 1, 2 ClyA, Rluc8

[0165] [Experimental Example 1] Evaluation of protein expression and activity of recombinant Salmonella

[0166] [1-1] Comparison of growth of Salmonella mutant strain and existing strain

[0167] The recombinant SHJ2037 and CNC18 colonies prepared in Comparative Example 1 and Preparation Example were grown overnight in LB liquid medium containing ampicillin, then diluted 1:100 with fresh LB medium and cultured further. When OD... 600 When the OD value reached 0.5 to 0.7, doxycycline diluted with ethanol to a final concentration of 200 ng / ml was added to the culture, and then the culture was incubated in a shaking incubator at 200 rpm and 37°C. The OD values ​​at different incubation time points were measured. 600 Value analysis of the growth pattern of the strain, the results are as follows Figure 2 As shown.

[0168] Meanwhile, existing, non-recombinant Salmonella colonies were treated in the same manner as described above, and the results were as follows: Figure 3 As shown.

[0169] like Figure 2 and Figure 3As shown, the recombinant Salmonella CNC18 exhibited almost no difference in growth rate compared to existing attenuated Salmonella SHJ2037 and wild-type Salmonella strains, and its growth was not particularly inhibited even during protein expression using doxycycline. Therefore, this confirms that the deletion of pathogenic genes in the constructed Salmonella strain does not affect the growth and gene expression of the Salmonella strain.

[0170] [1-2] Comparison of protein expression level by Western blot analysis

[0171] To compare the expression levels of the cytolysin A (ClyA) gene between the recombinant Salmonella strains SHJ2037 and CNC18 constructed in Comparative Example 1 and Preparation Example, the expression of the Rluc8 protein in the strains cultured in Experimental Example [1-1] was analyzed by Western blot analysis using a protein-specific antibody.

[0172] Specifically, the cultures of each strain cultured in Experimental Example [1-1] were diluted with PBS to a concentration of 4 × 10⁻⁶. 7 The concentration was set at CFU / ml, and the sample was centrifuged at 13000 rpm for 5 minutes, and the precipitate was collected. The precipitate was washed with PBS and mixed with SDS sample buffer containing 0.2% β-mercaptoethanol (catalog number EBA-1052.ELPIS BIOTECH) to obtain bacterial lysates. The bacterial lysates were then electrophoresed on a 12% SDS-PAGE gel, and the proteins on the gel were transferred to a nitrocellulose membrane and blocked with 5% skim milk at room temperature. The expression level of Rluc8 protein was then analyzed using an Rluc8 antibody (catalog number AB3256, Millipore, USA), and the results are shown below. Figure 4 As shown.

[0173] like Figure 4 As shown, it was confirmed that the two recombinant Salmonella strains SHJ2037 and CNC18 overexpressed the Rluc8 gene, and there was no significant difference in the Rluc8 gene expression level between the strains.

[0174] [ 1-3 Comparing protein functional expression levels using activity assays

[0175] To measure the luciferase activity in the strains cultured in Experimental Example 1, the strains were resuspended in 1 ml of PBS. Then, 1 μg / ml of the substrate coelenterate diluted in ethanol was added to the resuspended strains. Finally, an in vivo imaging system (Berthold Technologies, GmbH & Co. KG, Germany) or Biorad Imager ChemoDoc was used. TMThe XRS+ system was used to measure the luciferase activity in the strains at an exposure time of 1 second. The CFU values ​​for each strain were normalized, and the normalized values ​​were calculated as relative luminescent units (RLUs). The results are as follows: Figure 6 As shown.

[0176] like Figure 6 As shown, luciferase activity was only detected in the presence of doxycycline, and the susceptibility and maximum activity of doxycycline were similar among strains.

[0177] In addition, the recombinant Salmonella strain diluted with PBS selected in Preparation Example 3 was inoculated onto blood agar plates containing 0 or 20 ng / ml doxycycline and incubated overnight at 37°C. The plates were then imaged. The results are as follows: Figure 7 As shown.

[0178] like Figure 7 As shown, this confirms that the blood agar hemolytic activity of this strain only occurs when doxycycline (+) is present, which contains the gene encoding cytolysin A.

[0179] The above results confirm that the recombinant Salmonella strain CNC18 of the present invention can regulate the expression of cytolysin A (ClyA) gene and act as an anticancer gene vector.

[0180] [Experimental Example 2] Detection of DAMP signal released by tumor cells treated with recombinant Salmonella

[0181] To assess the effects of recombinant Salmonella strains on tumor cells, changes in damage-associated molecular patterns (DAMPs) that elicit an immune response in tumor cells were investigated when tumor cells were treated with SHJ2037 and CNC18 strains, respectively. Extracellular ATP release was measured within the DAMPs. Specifically, mouse CT26 colon cancer cell lines CRL-2638 and HB-8064 (ATCC, USA) were cultured in high-glucose DMEM (Dulbecco modified Eagle medium) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator, and then treated with recombinant Salmonella. ATP from each culture was measured at different time points using an assay kit, and the results are as follows: Figure 8 As shown.

[0182] like Figure 8 As shown, the increased release of ATP from tumor cells under Salmonella treatment indicates that the Salmonella strain of the present invention can be used for tumor treatment.

[0183] [Experimental Example 3] Distribution Analysis of Recombinant Salmonella in Small Animal Tumor Models

[0184] Attenuated Salmonella strains were injected into small animal mouse models for anti-cancer treatment research, and the growth rate and distribution of the strains in the host were examined.

[0185] Specifically, four bacterial strains (SHJ2037, CNC16, CNC17, and CNC18) were cultured in LB liquid medium, then washed and diluted with PBS. Each strain was injected into the tail vein of each mouse, resulting in a final bacterial concentration of 2 × 10⁻⁶. 7 CFU / mouse was used, and three mice from each strain were euthanized. Host organs were then extracted, minced, and diluted. Each dilution was plated and incubated on solid LB medium, and viable cells were counted. Results are as follows: Figure 9 As shown. Furthermore, mice carrying tumors were treated in the same manner as described above: the host tumors were extracted, minced, and diluted, plated on solid LB medium, and incubated. Viable cells were counted, and the results are shown below. Figure 10 As shown.

[0186] The results are as follows Figure 9 and Figure 10 As shown, strain CNC18 was confirmed to die rapidly in the liver, exhibiting very low viability in the liver, but remained in the tumor, indicating its specific and selective targeting ability.

[0187] On the other hand, such as Figure 9 As shown, strain CNC16 exhibits higher activity in the liver than strain SHJ2037, and at least 100 times higher activity than strain CNC18. This demonstrates that strain CNC16 possesses non-specific targeting capabilities in organs other than tumors.

[0188] Furthermore, CNC17 exhibited higher activity in the liver than strain SHJ2037, indicating that strain CNC17 also possesses non-specific targeting capabilities.

[0189] like Figure 10 As shown, the survival rate of strain CNC17 in tumors was at least 10 times lower than that of strain CNC18. In other words, it is confirmed that strain CNC17 is less effective against tumors than strain CNC18.

[0190] [Experimental Example 4] Analysis of high inflammatory response after Salmonella injection (1)

[0191] First, to detect the high inflammatory response after Salmonella injection, nine CT26 tumor model mice were injected with strains SHJ2037 and CNC18, and PBS (three mice per strain or PBS), respectively. Spleen weight was measured and compared at different time points. Figure 11 As shown, each mouse underwent laparotomy to remove its spleen, and the size of the spleen in each mouse was measured. The results are as follows: Figure 12 As shown.

[0192] like Figure 12 As shown, compared with the PBS-treated control group, the spleen size of the strain-treated group increased, but the spleen size of the strain CNC18-treated group was smaller than that of the strain SHJ2037-treated group, indicating that the Salmonella strain of the present invention minimizes high inflammatory response.

[0193] [Experimental Example 5] Analysis of high inflammatory response after Salmonella injection (2)

[0194] In addition, to examine the high inflammatory response after Salmonella injection, strains SHJ2037, CNC16, CNC17 and CNC18 were injected into mice in the same manner as described in Experimental Example 4, and the spleen weight of each mouse was measured and compared on days 1, 3 and 5.

[0195] As a result, Figure 13 As shown, the spleen weight of the groups treated with strains SHJ2037, CNC16, and CNC17 increased compared with the control group treated with PBS, but the spleen weight increase of CNC18 was the smallest among the strain-treated groups.

[0196] The above results confirm that the attenuated Salmonella strain of the present invention dies rapidly from the early stage of infection, and its activity in all normal organs is significantly reduced, while maintaining a strong tumor-targeting ability, indicating that the strain has minimal side effects on the host.

[0197] [Experimental Example 6] Analysis of the anticancer effect of attenuated Salmonella in small animal tumor models (1)

[0198] The tumor cells (CT26 1x10) cultured in Experimental Example 2 were used. 6 Tumor animal models were established by subcutaneous injection of cells / mouse into the flanks of BALB / c mice (n=7). Salmonella strains SHJ2037 and CNC18 were injected into the tail vein of each tumor animal model, respectively. To assess the anticancer effect of each strain in the tumor animal models, mice were anesthetized with 2% isoflurane, and tumor volume (mm²) was measured using the formula (length × height × width) / 2. 3 The result is as follows: Figure 14 and 15 As shown.

[0199] like Figure 14 and 15 As shown, compared with the control group, cancer growth in the Salmonella-treated group was inhibited, and the survival rate of the treated group was increased. In particular, strain CNC18 was shown to be significantly more effective than SHJ2037 in inhibiting tumor growth.

[0200] Therefore, it was confirmed that strain CNC18 dies rapidly from the initial stage of infection, its viability is significantly reduced in all normal organs, while maintaining a strong tumor-targeting ability, exhibiting excellent tumor treatment effects and minimizing side effects caused by host infection.

[0201] [Experimental Example 7] Analysis of the anticancer effect of attenuated Salmonella in small animal tumor models (2)

[0202] The cultured tumor cells (MC38, mouse colon adenocarcinoma cells, 1x10⁻¹) 6 Tumor animal models were established by subcutaneous injection of cells / mouse into the flank of mice (C57BL / 6, n=6). Salmonella strains SHJ2037 and CNC18 were injected into the tail vein of each tumor animal model, respectively. To assess the anticancer effect of each strain in the tumor animal models, mice were anesthetized with 2% isoflurane, and tumor volume (mm²) was measured using the formula (length × height × width) / 2. 3 The result is as follows: Figure 16 and 17 As shown.

[0203] like Figure 16 and 17 As shown, compared with the control group, cancer growth in the Salmonella-treated group was inhibited, and the survival rate of the treated group was increased. In particular, strain CNC18 was shown to be more effective than SHJ2037 in inhibiting tumor growth.

[0204] [Experimental Example 8] Evaluation of the immune activation effect of Salmonella injection

[0205] To evaluate the immune activation effect of Salmonella injection, immune cells were measured and compared. Salmonella strains SHJ2037 and CNC18 were injected into the tumor animal model constructed in Experimental Example 6, and immune cells were collected on day 3. Immune cells were collected from the tumor and surrounding lymph nodes. The amount of collected immune cells was measured, and the results are shown in... Figure 18 to Figure 24 middle.

[0206] like Figure 18 to 22As shown, compared with the PBS control group and the SHJ2037 control group, the CNC18 treatment group showed an increase in the proportion of leukocytes, neutrophils, natural killer cells, and CD8+ T cells, and an increase in the proportion of dendritic cells in the lymph nodes. On the other hand, as Figure 23 and 24 As shown, compared with the PBS control group and the SHJ2037 control group, the proportion of immunosuppressive M2 macrophages and regulatory T (Treg) cells was reduced in the CNC18 treatment group.

[0207] The above results confirm that the attenuated Salmonella strain CNC18 of this invention has a significantly enhanced immune function compared with the PBS control group and the SHJ2037 control group.

[0208] [Experimental Example 9] Salmonella Tumor Imaging Analysis (1)

[0209] Tumor cells cultured as described in Experimental Example 2 were subcutaneously injected into the flanks of mice to construct a tumor animal model. Salmonella strains SHJ2037lux and CNC18lux were injected into these models, respectively. Here, strains SHJ2037lux and CNC18lux are strains in which a luminescent gene has been introduced into strains SHJ2037 and CNC18, respectively. The bacterial luciferase gene (lux) was used as the luminescent gene.

[0210] To evaluate the imaging performance of each strain in tumor animal models, organs and tumors were extracted at each time point and then imaged using an in vivo imaging system (IVIS). Results are as follows: Figure 25 to 27 As shown.

[0211] like Figure 25 to 27 As shown, it was confirmed that, similar to strain SHJ2037lux, when strain CNC18lux was injected, it was able to perform real-time imaging, while simultaneously displaying tumor-specific and selective activity.

[0212] [Experimental Example 10] Salmonella Tumor Imaging Analysis (2)

[0213] A multiple myeloma model was established by injecting cultured MOPC cells into the tibia of mice. Salmonella strains SHJ2037lux and CNC18lux were then injected into the model, respectively, in the same manner described in Experimental Example 9. The mice were then imaged at each time point using an in vivo imaging system (IVIS), and the results are as follows: Figure 28 and 29 As shown.

[0214] like Figure 28 and 29As shown, the injected strain CNC18lux was able to perform real-time imaging, simultaneously displaying tumor-specific and selective activity. Specifically, unlike strain SHJ2037lux, which displays scattered image signals around the tumor, strain CNC18lux reached deeper layers of the tumor, thus achieving clearer real-time imaging.

[0215] Experimental Examples 1 to 10 demonstrate that the CNC18 strain dies rapidly from the initial stage of infection, exhibits significantly reduced viability in all normal organs, maintains strong tumor-targeting ability, and demonstrates excellent tumor treatment effects while minimizing side effects caused by host infection.

[0216] Although the invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is merely a preferred embodiment and does not limit the scope of protection of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0217] Industrial applicability

[0218] This invention relates to a Salmonella strain that selectively targets cancer and a composition containing the strain for the prevention or treatment of cancer. sequence list <110> Chonnam National University Industry-Academia Collaboration Group <120> Salmonella strains for cancer treatment and their applications <130> POPB204121PCT <150> KR 10-2020-0074197 <151> 2020-06-18 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 35012 <212> DNA <213> unknown <220> <223> Salmonella pathogenicity island-1 <400> 1 taattatatc atgatgagtt cagccaacgg tgatatggcc ttataaggct tgcagtcttt 60 catgggcagc aagtaacgtc tgatatatgc ttaaattctt acttccgggt tcaagcaaaa 120 cttttttaaa ttcggtcatg agttgctctt catcttcttt cgaacgcatg tattgtggat 180 gttcctggaa gaaggtgagc gcctgttctt tggtttgttt atatttttcg caaaaaatgc 240 ttgagctgat tgcgctattt tttgatgcgg aattatcagc gttatggttt gataatgatt 300 tattcttgc aaggtctgac ggcacatacg gaagagactg acactcatca atactatttg 360 cgttggccag ttgctctttc tgagcgccag gttgctgtac cggtttgctc acggaggaag 420 ggaggggcac ctgagcacag ccgatcagta aaaagacagg aagacagcta taaaattttt 480 tcatattaac tccaactaca gaagaatgag gcgccatgtt tttacacaac cgtcagaacg 540 ttatctgaca atagttataa gggggcgaat gcgtttcagg aatgctagca aaaaaaaactc 600 ctgatataaa taattaattc tccatccaga tgacaatatc tgaatttttg ctgctgcaga 660 acaataagcc agaatttata cactatgagc taattttcta ttttaaatca gcaacacta 720 tgcaagttgg ccaatttaat tagcgggcgg catcagtttc ataatgattg catcaggatt 780 ttgccacccg ctcccggtat tgtttacata ttaaaatgat ttttaactgg tgctgacaac 840 tatgctaaat acgcaggaag tacttaaaga aggagagaag cggaaaatcc gcagcccgga 900 agcatggttt atacagacgt gttccgcgca aaagctgcat atgtcatttt ctgaaagccg 960 acacaatgaa aattgcctga ttcaggaagg cgcgctgctt ttttgcgagc aggccgttgt 1020 cgcaccagta tcaggagacc tggtttttcg accgttaaaa attgaagtac tcagcaaatt 1080 actggcattt atcgatggcg caggattagt ggacacgaca tatgctgaat ccgataaatg 1140 ggttttgctg agtcctgagt ttcgcgctat ttggcaagat cgtaaacgct gcgagtactg 1200 gtttttgcag caaattatta cgcttctcc ggccttcaat aaggtactgg cgctgttacg 1260 aaaaagcgag agttactggt tggttggcta tttactcgct cagtcaacca gcggcaacac 1320 gatgagaatg ctgggagaag actatggcgt ttcttatacc cattttcgtc gtttgtgcag 1380 cagagcgttg ggcggaaaag cgaagagtga attacgaaac tggcgtatgg cgcaatcgct 1440 gctgaatagt gtagaaggcc acgagaacat cacccaatta gccgttaatc atggttactc 1500 atcgccttca catttttcta gtgagatcaa agagctgatc ggcgtttcgc cgcggaaatt 1560 atcaaatatt attcaattgg cagacaaatg aagacacata ttcttttggc cagagtgctg 1620 gcatgtgccg cgcttgttct ggttacacct ggttattcta gtgaaaaaat acctgtaacg 1680 ggaagtgggt ttgttgcgaa agacgatagc ctgcggacat ttttcgatgc catggcgcta 1740 cagctaaagg agcctgtcat tgttagcaaa atggcggcac gaaaaaaaat tacgggcaac 1800 tttgagtttc acgatcctaa cgcattactg gagaagcttt ccctacaact ggggctgatt 1860 tggtatttcg atgggcaggc tatctatatt tatgacgcca gtgaaatgcg caatgccgtg 1920 gtttctttac gcaacgtctc actcaatgag ttcaacaatt ttctaaaacg ctcaggttta 1980 tataacaaaa attacccgct acgtggcgat aaccgtaaag gaacattcta tgtttcaggg 2040 ccacccgtct atgttgatat ggtggtcaac gccgccacca tgatggacaa gcaaaacgat 2100 ggtattgagc tgggacgcca gaaaataggg gtgatgcgtc tgaacaatac cttcgtgggc 2160 gatcgtacct ataatctgcg cgatcagaaa atggttattc ccggtattgc tacggccatt 2220 gaaaggttat tgcagggaga agagcaaccc ttaggtaata ttgtcagtag cgaacccccg 2280 gcgatgccag cgttttcagc gaatggagaa aaaggtaaag cagcaaatta tgccggtggc 2340 atgagtctgc aggaagcttt aaagcaaaat gccgcggcgg gcaatattaa aatcgtggcc 2400 tatccggata ccaacagttt gttagtaaag ggaacggctg agcaggtgca ttttatcgaa 2460 atgctggtta aagcgctgga tgtcgccaaa cgtcacgtag aattatccct gtggattgtc 2520 gatcttaata aaagcgatct ggagcgtttg ggcacttcat ggagcggcag cataactatt 2580 ggggacaaac ttggcgtgtc attaaaccag tcttcaataa gtaccctcga tggcagtcga 2640 ttcatcgccg cggtcaatgc gttagaagag aagaaacagg cgacggtggt ttcgcgcccg 2700 gtattactga ctcaggaaaa tgttcccgct atttttgata acaacagaac gttttacacc 2760 aagctgattg gggaacgtaa tgtggcgctt gagcatgtaa catacggaac aatgatccga 2820 gtgctgcccc gtttttccgc agatggtcag atagaaatgt cgctggacat tgaagatggc 2880 aacgataaga cgccgcaatc tgatactacc acctccgtag atgcgttacc cgaagtcggg 2940 cgaacgttaa ttagcactat tgcgagagtg ccgcacggaa aaagtttgct ggtcgggggt 3000 tatacacggg atgcaaatac cgatactgtc caaagtattc cgtttttagg caaattaccg 3060 cttattggta gcctgttccg ttattccagt aagaataaaa gtaatgttgt tcgtgtgttc 3120 atgattgaac caaaagaaat tgtcgacccg ttaacgccgg atgccagcga atcggtaaac 3180 aatattctga agcaaagcgg tgcctggagt ggggacgata agttacagaa atgggttcgt 3240 gtttatctgg atagaggtca ggaggcaatt aaatgattcc tggctcaacc tccggtattt 3300 cattttccag aatattgtcc cggcagacat ctcatcagga tgcgacccag catactgatg 3360 cgcaacaggc ggaaatacaa caggccgcag aggattcgtc tccaggggcg gaagtacaaa 3420 aatttgtcca gtcgacggac gaaatgtcag cggcgctggc gcaatttcgt aaccgtcgcg 3480 attatgaaaa aaaatccagt aatttatcta acagttttga acgcgtgctg gaggatgagg 3540 ctttaccgaa ggcgaagcaa atcttaaagc taattagcgt acatggcggc gcgttagaag 3600 atttttacg tcaggcgcgt agcttatttc ctgaccccag tgatttagtc cttgtgttac 3660 gcgaattgct tcgtcgtaaa gacctggaag agatcgtgcg gaaaaagctg gagtcgttac 3720 ttaagcacgt tgaagagcaa accgatccga agaccctcaa ggcagggatt aattgtgcgt 3780 tgaaggcccg gctttttggg aaaacattat cgttaaaacc aggcttattg cgcgccagct 3840 atcggcaatt tatccagagt gaatcacatg aagtggagat ttactctgac tggatagcca 3900 gttatggcta tcaacgtcga ctggtggtac tggattttat tgagggttcg ctattaaccg 3960 atattgacgc gaatgacgcc agctgttcgc gcctggagtt tggccagctt ttacgacgcc 4020 tgacgcaact taaaatgttg cgctccgctg acctactgtt tgtgagtaca ttgttgtcgt 4080 attcgtttac caaagcgttt aatgcggagg agtcgtcgtg gttactactg atgctttcgc 4140 tattgcaaca gccacatgaa gtggattcgc tgttagccga tattataggt ttgaatgcgt 4200 tattgcttag tcataaagaa catgcatcct ttttgcagat attttatcaa gtatgtaaag 4260 ccataccctc ttcactcttt tatgaagaat attggcagga agaattgtta atggcgttac 4320 gtagtatgac cgatattgcc tacaagcatg aaatggcaga acagcgtcgt actattgaaa 4380 agctgtctta atttaatatt aacaggatac ctatagtgct gctttctcta cttaacagtg 4440 ctcgtttacg acctgaatta ctgattctgg tactaatggt gatgatcatt tctatgttcg 4500 tcattccatt acctacctat ctggttgatt tcctgatcgc actgaatatc gtactggcga 4560 tattggtgtt tatggggtcg ttctacattg acagaatcct cagtttttca acgtttcctg 4620 cggtactgtt aattaccacg ctctttcgtc tggcattatc gatcagtacc agtcgtctta 4680 tcttgattga agccgatgcc ggtgaaatta tcgccacgtt cgggcaattc gttattggcg 4740 atagcctggc ggtgggtttt gttgtcttct ctattgtcac cgtggtccag tttatcgtta 4800 ttaccaaagg ttcagaacgt gtcgcggaag tcgcggcccg attttctctg gatggtatgc 4860 ccggtaaaca gatgagtatt gatgccgatt tgaaggccgg tattattgat gcggatgccg 4920 cgcgcgaacg gcgaagcgta ctggaaaggg aaagccagct ttacggttcc tttgacggtg 4980 cgatgaagtt tatcaaaggt gacgctattg ccggcatcat tattatcttt gtgaacttta 5040 ttggcggtat ttcggtgggg atgactcgcc atggtatgga tttgtcctcc gccctgtcta 5100 cttataccat gctgaccatt ggtgatggtc ttgtcgccca gatccccgca ttgttgattg 5160 cgattagtgc cggttttatc gtgacccgcg taaatggcga tagcgataat atggggcgga 5220 atatcatgac gcagctgttg aacaacccat ttgtattggt tgttacggct attttgacca 5280 tttcaatggg aactctgccg ggattcccac tgccggtttt tgttatttta tcggtggttt 5340 taagcgtact cttctatttt aaattccgtg aagcaaaacg tagcgccgcc aaacctaaaa 5400 ccagcaaagg cgagcagccg ctcagtattg aggaaaaaga agggtcgtcg ttaggactga 5460 ttggcgatct cgataaagtc tctacagaga ccgtaccgtt gatattactt gtgccgaaga 5520 gccggcgtga agatctggaa aaagctcaac ttgcggagcg tctacgtagt cagttcttta 5580 ttgattatgg cgtgcgcctg ccggaagtat tgttacgaga tggcgagggc ctggacgata 5640 acagcatcgt attgttgatt aatgagatcc gtgttgaaca atttacggtc tattttgatt 5700 tgatgcgagt ggtaaattat tccgatgaag tcgtgtcctt tggtattaat ccaacaatcc 5760 atcagcaagg tagcagtcag tatttctggg taacgcatga agagggggag aaactccggg 5820 agcttggcta tgtgttgcgg aacgcgcttg atgagcttta ccactgtctg gcggtgacgc 5880 tggcgcgcaa cgtcaatgaa tatttcggta ttcaggaaac aaaacatatg ctggaccaac 5940 tggaagcgaa atttcctgat ttacttaaag aagtgctcag acatgccacg gtacaacgta 6000 tatctgaagt tttgcagcgt ttgttaagcg aacgtgtttc cgtgcgtaat atgaagttaa 6060 ttatggaagc gctcgcattg tgggcgccaa gagaaaaaga tgtcattaac cttgtggagc 6120 atattcgtgg agcaatggcg cgttatattt gtcataaatt cgccaatggc ggcgaattac 6180 gagcagtaat ggtatctgct gaagttgagg atgttattcg caaagggatc cgtcagacct 6240 ctggcagtac cttcctcagc cttgacccgg aagcctccgc taatttgatg gatctcatta 6300 cacttaagtt ggatgattta ttgattgcac ataaagatct tgtcctcctt acgtctgtcg 6360 atgtccgtcg atttattaag aaaatgattg aaggtcgttt tccggatctg gaggttttat 6420 ctttcggtga gatagcagat agcaagtcag tgaatgttat aaaaacaata taagggctta 6480 attaaggaaa agatctatgc aacatttgga tatcgctgaa ttagttcgtt ccgcactgga 6540 agtaagtggt tgcgatcctt cattaatttgg aggaatagat agccattca cattgttct 6600 ggatttattt gcattgccaa gtatctgtat cagcgtcag gacgatgatg tatggatctg 6660 ggcgcaattg gtgctgaca gcatggtggt attackacag cgggcttatg aaatcttaat 6720 gaccataatg gaaggatgcc atttgcccg cggcgggcaa ttactactgg gggagcagaa 6780 tggggagcta acgcttaaag ccttagtgca tccggatttt ttatctgacg gtgaaaagtt 6840 ctctactgcc ttgaatgggt tttacacta tctggagtt tttagtcggt cgctaatgg 6900 atgaaaacac ctcgtttact gcaatactctg gcctaccac aaaaaataac cggcccaatt 6960 attgaggcgg aattgcgcga tgtggccatt ggcgaactgt gtgaaatacg ccgtggctgg 7020 caccaaaaac aggttgttgc acgtgcgcag gtggttggct tacagcggga acgcaccgtg 7080 ctgagcctta tcggcaatgc ccaggggctg agccgcgatg tcgttta tcccactgga 7140 cgtgcgttat cggcgtgggt gggatactcg gtattaggcg cggtgttgga tccgacaggg 7200 aaaatcgttg agcgttttac ccctgaagtg gcgccgatta gcgaagaacg cgttattgat 7260 gtcgcaccgc cgtcttacgc ttcacgcgtt ggcgtccgtg aaccgctgat taccggtgtg 7320 cgcgcgattg acgggttatt gacctgtggc gtaggccagc gaatgggcat ttttgcctcc 7380 gcaggatgcg gtaagaccat gctgatgcat atgctgatcg agcaaacgga ggcggatgtc 7440 tttgttatcg gtcttatcgg tgaacgaggc cgtgaggtca ctgaattcgt ggatatgttg 7500 cgcgcttcgc ataagaaaga aaaatgcgtg ctggtttttg ccacttccga tttcccctcg 7560 gtcgatcgct gcaatgcggc gcaactggcg acaaccgtag cggaatattt tcgcgaccag 7620 ggaaaacggg tcgtgctttt tatcgattcc atgacccgtt atgcgcgtgc tttgcgagac 7680 gtggcactgg cgtcgggaga gcgtccggct cgtcgaggtt atcccgcctc cgtattcgat 7740 aatttgcccc gcttgctgga acgcccaggg gcgaccagcg agggaagcat tactgccttt 7800 tatacggtac tgctggaaag cgaggaagag gcggacccga tggcggatga aattcgctct 7860 atccttgacg gtcacctgta tctgagcaga aagctggccg ggcagggaca ttacccggca 7920 atcgatgtac tgaaaagcgt aagccgcgtt tttggacaag tcacgacgcc gacacatgct 7980 gaacaggcat ctgccgtgcg taaattaatg acgcgtttgg aagagctcca gcttttcatt 8040 gacttgggag aatatcgtcc tggcgaaaat atcgataacg atcgggcgat gcagatgcgg 8100 gatagcctga aagcctggtt atgccagccg gtagcgcagt attcatcctt tgatgacacg 8160 ttgagcggta tgaatgcatt cgctgaccag attaaagta ttgcagcggc gctgtacggt 8220 atttcattca cagtgtgagt cgatattact tcgctatcag gatgaggacc gcgggctgca 8280 ggccgaggag gaggcgatcc ttgaacaaat agcgggtctg aaattgttat tagatacgct 8340 gcgtgcagaa aacagacagc tcagtcgtga ggaaatttat acgttattac gtaagcagtc 8400 tattgttcgc cggcagataa aagatttaga actccagatt atacaaattc aggaaaaacg 8460 gagcgagctg gaaaagaaaa gggaagagtt tcagaaaaaa agtaaatatt ggttgcgcaa 8520 agaagggaac tatcaacgct ggataatccg tcagaaaaga ttctatatcc agcgagagat 8580 acagcaggaa gaggccgagt cagaggagat aatttaatgg gcgatgtgtc agctgtcagt 8640 tcatccggga acattttact gccgcagcag gatgaggttg gcggtttatc agaagcatta 8700 aaaaaagcgg tggaaaaca tagcaga tattccggtg ataaaaaga tcgcgactat ggcgatgctt tcgtaatgca taaagaaacg gctttaccgt tattactggc ggcatggcga catggcgcgc cagcgaatc aagaacatcac aatggcaacg tttctggtct gcatcatac 8940. ggaaaaagcg aactcaggat tgctgaaaaa ctgttgaaag tcactgctga aaaatctgtc ggtttgatct ctgcggaggc caaagtagt aaatccgcag cgttgctatc gtctaaaaat aggccgttag aaagcgtag cggtaaaaaa ttatctgctg atttaaaagc tgtggaatcc gttagtgaag taaccgataa cgccacggga atctctgacg father ggcattgcct ggggataata aagccatcgc gggcgaaggc gttcgtaaag agggcgcgcc gctggcgcgg 9180 gatgtcgcac ctgcccgaat ggccgcagcc aataccggta agcctgaaga taaagatcat aaaaaggtta aagatgtttc tcagcttccg ctgcaaccaa ccactatcgc cgatcttagc caattaaccg gcggcgatga aaaaatgcct ttagcggcgc aatcaaagcc gatgatgact atttttccca ctgccgatgg cgtgaaagga gaggatagct cgctgactta ccgttttcag cgctgggga atgactattc cgtcaatatt caggcgcggc aagcagggga gttttcgtta ataccgtcaa atacgcaggt tgaacatcgt ttgcatgatc aatggcaaaa cggtaatccc cagcgctggc acctgacgcg agcgatcaa caaaatccgc agcagcaaca gcacagacag caatctggcg aggaggatga cgcctgatgt cattgcgtgt gagacagatt gatcgtcgcg aatggctatt ggcgcaaacc gcgacagaat gccagcgcca tggccggga gcgacgctgg aatatccgac gcgacaggga atgtgggttc ggttgagcga tgcagaaaaa cggtggtcgg 9780. cctggattaa acctggggac tggcttgagc atgtctctcc cgctctggct ggggcggcgg tttctgctgg cgctgagcac ctggtcgttc cctggcttgc tgcaacagag cgaccgtttg 9900 agttgcccgt gccgcatttg tcctgtcggc gtttatgcgt agagaacccc gtaccggga gcgcgctgcc ggagggaa ttgttgcaca ttatgagcga tcggggcggc ctgtggtttg agcatcttcc tgaactgcct gcagtcgggg gcggcaggcc gaaaatgctg cgttggccgt tgcgctttgt aatcggtagc agtgatacgc agcgttcgtt gctgggccga atcgggatcg 10140 gagatgtact cctgattcgt acttcccgtg cggaagttta ttgctacgcg aaaaagttag 10200 gtcatttcaa ccgtgttgaa gggggaatta ttgtggaaac gttagatatt caacatatcg 10260 aagaagaaaa taatacaact gaaactgcag aaactctgcc tggcttgaat caattgcccg 10320 tcaaactgga atttgttttg tatcgtaaga acgttaccct cgccgaactc gaagccatgg 10380 ggcagcaaca gctattatca ctgccgacca atgctgaact taacgttgaa attatggcga 10440 atggtgtttt gctgggtaat ggcgaactgg tacagatgaa tgacacctta ggcgttgaga 10500 tccatgaatg gctgagcgag tctggtaatg gggaatgata tctcattaat tgccttactg 10560 gcattttcca ccctgttgcc atttattatt gcgtcaggaa cctgtttcgt taaattttct 10620 attgtatttg tcatggtgcg taacgccctg ggattacagc agataccttc aaatatgacg 10680 cttaacggcg tcgcattgct gctttctatg tttgttatgt ggcccataat gcatgatgcc 10740 tacgtctatt ttgaggacga agatgtcacc tttaatgata tttcatcatt aagtaaacac 10800 gttgatgaag gtctggatgg ttatcgcgat tatctgatca aatattcaga tcgcgagtta 10860 gttcagtttt ttgaaaacgc gcaactgaag cgtcagtatg gagaagagac cgagacggta 10920 aagcgtgaca aagatgaaat tgaaaaacct tcaatatttg cgttattacc tgcttatgcg 10980 ctgagcgaaa taaaaagcgc gtttaaaatt ggtttttatc tttatttgcc ctttgtcgtc 11040 gtcgacctgg tggtatccag cgtgctactg gcgctgggga tgatgatgat gagtccggtg 11100 acgatatcta cacctattaa gctggtgctt tttgtcgcgc ttgatggctg gaccttactg 11160 tctaagggat tgatattaca gtatatggac attgcaacat gacatcatta cgagacggga 11220 tagttaaatg gatgatttag tgtttgcagg taataaggcg ctctatcttg ttttgatcct 11280 gtcagggtgg ccgacgattg tcgcaacgat tatcggcctc ctggtagggt tattccagac 11340 ggtaacgcaa ttacaggaac agacgctgcc ttttggcatt aaattacttg gcgtgtgttt 11400 atgcttgttt ttactgtctg gctggtatgg cgaagtttta ctctcttacg ggcgtcaggt 11460 gatattcctg gcgttggcta aggggtaaaa aatgttttac gcgttgtact ttgaaattca 11520 tcacctggtt gcgtctgcgg cgctagggtt tgctcgcgtg gcgccgattt ttttcttcct 11580 gccgtttttg aatagcgggg tattaagcgg tgcgccgaga aacgccatta tcatcctggt 11640 ggcattggga gtatggccgc atgcattgaa cgaggcgccg ccgtttttat cggtggcgat 11700 gatcccgtta gttctgcaag aagcggcggt aggcgtcatg ctgggctgtc tgctgtcatg 11760 gcctttttgg gttatgcatg cgctgggttg tattatcgat aaccagcgag gggcaacgct 11820 aagtagtagt atcgatccgg caaacggtat tgatacctcg gaaatggcta atttcctgaa 11880 tatgtttgcc gctgtcgttt atttacaaaa cggcggtctg gtcacgatgg ttgacgtgtt 11940 aaataaaagc tatcagctat gcgatccgat gaacgagtgc acgccttcat taccgccgct 12000 attaacgttt attaatcagg tggctcaaaa cgccttggtt ctggccagtc cggtggtatt 12060 agtgctgttg ctgtcagaag tattcctggg tttattgtcg cgctttgctc cgcaaatgaa 12120 cgcttttgcg atttcactga cggtaaaaag cggtattgcc gttttaatta tgctgcttta 12180 tttctctccg gtactaccgg acaatgtact gcgactctct ttccaggcca cagggttaag 12240 cagttggttt tacgagcgag gggcgacgca tgtcctcgaa taaaacagaa aaaccgacta 12300 aaaaacggct ggaagactcc gctaaaaaag gccagtcatt taaaagtaaa gatctcatta 12360 tcgcctgcct gacgctggga ggaattgcct atctggtgtc gtatggctca tttaatgagt 12420 ttatggggat aattaagatc attattgcgg ataatttga tcagagcatg gctgactaca 12480 gtttggccgt ttttgggata gggttaaaat atctgattcc attatgctg ctctgcttag 12540 tgtgttccgc attaccggcg ttattacagg ccggttttgt gctggcgaca gaagcattaa 12600 agcctaattt atcggcgtta aacccggtag aaggggcaaa aaaacttttt agtatgcgca 12660 cggttaaaga tacggtcaaa accctactgt atctctcatc cttttgtggtg gccgccatca 12720 tttgctggaa gaaatataag gttgaaatct tttctcagct aaatggcaat attgtaggta 12780 ttgccgtcat ttggcgtgaa cttctcctcg cattggtatt aacttgcctt gcttgcgcat 12840 tgattgtctt attattggat gctattgcgg aatatttcct gaccatgaaa gatatgaaaa 12900 tggataagga agaagtgaag cgtgaaatga aggagcagga agggaaccca gaggttaaat 12960 ctaaaagacg tgaagttcat atggaaattc tgtctgaaca ggtgaaatct gatattgaaa 13020 actcacgcct gattgttgcc aaccccacgc atattacgat cgggatttat tttaaacccg 13080 aattgatgcc gattccgatg atctcggtgt atgaaacgaa tcagcgcgca ctggccgtcc 13140 gcgcctatgc ggagaaggtt ggcgtacctg tgatcgtcga tatcaaactg gcgcgcagtc 13200 ttttcaaaac ccatcgccgt tatgatctgg tgagtctgga agaaattgat gaagttttac 13260 gtcttctggt ttggctggaa gaggtagaaa acgcgggcaa agacgttatt cagccacaag 13320 aaaacgaggt acggcattga gccgcgtaag gcagtagcga tgtattcatt gggcgttttt 13380 tgaatgttca ctaaccaccg tcggggttta ataactgcat cagataaacg cagtcgttaa 13440 gttctacaaa gtcggtgaca gataacagga gtaagtaatg gattatcaaa ataatgtcag 13500 cgaagaacgt gttgcggaaa tgatttggga tgccgttagt gaaggcgcca cgctaaaaga 13560 cgttcatggg atccctcaag atatgatgga cggtttatat gctcatgctt atgagtttta 13620 taaccaggga cgactggatg aagctgagac gttctttcgt ttcttgca tttatgatt 13680 ttacaatccc gattacacca tgggactggc ggcagtagc caacgaaa aacaatttca 13740 gaaagcatgt gacctttatg cagtagcgtt tacgttactt aaaaatgatt atcgccccgt 13800 ttttttacc gggcagtgtc aattattaat gcgtaggca gcaaagccazgagtgttt 13860 tgaacttgtc aatgaacgta ctgaagatga gtctctgcgg gcaaagcgt tggtctatct 13920 ggaggcgcta aaacggcgg agacagagca gcacagtgaa caagaaagg ataatttag 13980 gtaaatgacg caagtagcat tagccgtagc ggatataccc aaaatccgcg cctcgctgag 14040 gcggctttg aaggcgttcg taagacacg gactttaa aagcggcgga taagctttt 14100 aagatgtgg tggcaacgaa agccggcgac cttaagccg gacaagctc cggcgagagc 14160 gctattata cggtgggtct aaagccgcct acggacccg cccgggaaaa actctccagc 14220 gaagggcaat tgacattact gcttggcaag ttaatgaccc tactggggcga tgtttcgctg 14280 tctcaactgg agtctcgtct ggcggtatgg caggcgatga ttgagtcaca aaaagagatg 14340 gggattcagg tatcgaaga attccagacg gctctgggag aggctcagga ggcgacggat 14400 ctctatgaag ccagtatcaa aaagacggat accgccaaga gtgtttatga cgctgcgacc 14460 aaaaaactga cgcaggcgca aaaataattg caatcgctgg acccggctga ccccggctat 14520 gcacaagctg aagccgcggt agaacaggcc ggaaaagaag cgacagaggc gaagaggcc 14580 ttagataagg ccacggatgc gacggttaaa gcaggcacag acgccaaagc gaaagccgag 14640 aaagcggata acattctgac caaattccag ggaacggcta atgccgcctc tcagaatcag 14700 gtttcccagg gtgagcagga taatctgtca aatgtcgccc gcctcactat gctcatggcc 14760 atgtttattg agattgtggg caaaaatacg gaaaagcc tgcaaaacga tcttgcgctt 14820 ttcaacgcct tgcaaggaagg gcgtcaggcg gagatggaaa agaatcggcg tgaattccag 14880 gaagacacgc gcaaagccga ggaaacgaac cgcattatgg gatgtatcgg gaagtcctc 14940 ggcgcgctgc taaccattgt cagcgttgtg gccgctgttt ttaccggtgg ggcgagtctg 15000 gcgctggctg cggtgggact tgcggtaatg gtggccgatg aaattgtgaa ggcggcgacg 15060 ggagtgtcgt ttattcagca ggcgctaaac ccgattatgg agcatgtgct gaagccgtta 15120 atggagctga ttggcaaggc gattaccaaa gcgctggaag gattaggcgt cgataagaaa 15180 acggcagaga tggccggcag cattgttggt gcgattgtcg ccgctattgc catggtggcg 15240 gtcattgtgg tggtcgcagt tgtcgggaaa ggcgcggcgg cgaaactggg taacgcgctg 15300 agcaaaatga tgggcgaaac gattaagaag ttggtgccta acgtgctgaa acagttggcg 15360 caaaacggca gcaaactctt tacccagggg atgcaacgta ttactagcgg tctgggtaat 15420 gtgggtagca agatgggcct gcaaacgaat gccttaagta aagagctggt aggtaatacc 15480 ctaaataaag tggcgttggg catggaagtc acgaataccg cagcccagtc agccggtggt 15540 gttgccgagg gcgtatttat taaaaatgcc agcgaggcgc ttgctgattt tatgctcgcc 15600 cgttttgcca tggatcagat tcagcagtgg cttaaacaat ccgtagaaat atttggtgaa 15660 aaccagaagg taacggcgga actgcaaaaa gccatgtcttt ctgcggtaca gcaaaatgcg 15720 gatgcttcgc gttttattct gcgccagagt cgcgcataaa aactgccaaa ataaagggag 15780 aaaaatatgt taattagtaa tgtgggaata aatcccgccg cttatttaaa taatcattct 15840 gttgagaata gttcacagac agcttcgcaa tccgttagcg ctaaagatat tctgaatagt 15900 attggtatta gcagcagtaa agtcagtgac ctggggttga gtcctacact gagcgcgcct 15960 gcgccagggg tattaacgca aacccccgga acgatcacgt cctttttaaa agccagtatt 16020 caaaataccg acatgaatca ggatttgaat gctctggcaa ataatgtcac gactaaagcg 16080 aatgaggttg tgcaaaccca gttacgcgag cagcaggcag aagtcggaaa gttttttgat 16140 attagcggaa tgtcttccag tgccgttgcg ctgttggctg ccgcgaatac gttaatgctg 16200 acgttgaacc aggctgatag caaactgtct ggtaagttgt cattagtcag ttttgatgca 16260 gctaaaacga cggcaagctc catgatgcgc gaagggatga atgcgttgtc cggtagtatt 16320 tcccagagcg cgcttcagtt ggggatcact ggcgtgggcg ccaaactgga atataagggg 16380 ctgcagaatg aaagaggcgc gcttaaacat aatgccgcga agatcgataa actgaccact 16440 gaaagccaca gtattaaaaa cgtgctgaac gggcagaata gcgtcaaact cggtgctgaa 16500 ggcgtcgatt ctctgaaatc gttaaatatg aagaaaaccg gtaccgatgc gacgaaaaat 16560 cttaatgatg cgacgcttaa atctaatgcc ggaaccagcg ccacggaaag tctgggtatt 16620 aaagacagta ataaacaaat ctcccctgaa catcaggcta ttctgtcgaa acgtcttgag 16680 tctgtcgaat ccgatattcg tcttgagcag aataccatgg atatgacccg aatcgatgcg 16740 cgcaagatgc agatgacggg cgatctgatt atgaagaact cggtcacggt cggtggtatt 16800 gcaggggcgt ccgggcagta cgccgctact caggaacgtt ccgagcagca aattagccag 16860 gtgaataacc gggttgccag caccgcatcg gacgaagccc gtgaaagttc acgtaaatcg 16920 accagcctga ttcaggaaat gctgaaaaca atggagagca ttaaccagtc gaaagcatcc 16980 gcactcgctg ctatcgcagg caatattcgc gcttaatctg aaaggtcatc tatacgccat 17040 catgggtgtg atttaatcgc gctcctgatg gcgaactggg gatattatgc ttaatattca 17100 aaattattcc gcttctcctc atccggggat cgttgccgaa cggccgcaga ctccctcggc 17160 gagcgagcac gtcgagactg ccgtggtacc gtctaccaca gaacatcgcg gtacagatat 17220 catttcatta tcgcaggcgg ctactaaaat ccaccaggca cagcagacgc tgcagtcaac 17280 gccaccgatc tctgaagaga ataatgacga gcgcacgctg gcgcgccagc agttgaccag 17340 cagcctgaat gcgctggcga agtccggcgt gtcattatcc gcagaacaaa atgagaacct 17400 gcggagcgcg ttttctgcgc cgacgtcggc cttatttagc gcttcgccta tggcgcagcc 17460 gagaacaacc atttctgatg ctgagatttg ggatatggtt tcccaaaata tatcggcgat 17520 aggtgacagc tatctgggcg tttatgaaaa cgttgtcgca gtctataccg atttttatca 17580 ggccttcagt gatattcttt ccaaaatggg aggctggtta ttaccaggta aggacggtaa 17640 taccgttaag ctagatgtta cctcactcaa aaatgattta aacagtttag tcaataaata 17700 taatcaaata aacagtaata ccgttttatt tccagcgcag tcaggcagcg gcgttaaagt 17760 agccactgaa gcggaagcga gacagtggct cagtgaattg aatttaccga atagctgcct 17820 gaaatcttat ggatccggtt atgtcgtcac cgttgatctg acgccattac aaaaaatggt 17880 tcaggatatt gatggtttag gcgcgccggg aaaagactca aaactcgaaa tggataacgc 17940 caaatatcaa gcctggcagt cgggttttaa agcgcaggaa gaaaatatga aaaccacatt 18000 acagacgctg acgcaaaaat atagcaatgc caattcattg tacgacaacc tggtaaaagt 18060 gctgagcagt acgataagta gcagcctgga aaccgccaaa agcttcctgc aaggataaca 18120 gaagaggata ttaataatgg ttacaagtgt aaggactcag ccccccgtca taatgccagg 18180 tatgcagacc gagatcaaaa cgcaggccac gaatctttgcg gcgaatcttt ccgcagtcag 18240 agaaagtgcc acagcgacgc tgtcagggga attaaaggc ccgcaactgg aagattttcc 18300 cgcgctgatc aaacaggcga gtctggatgc attgtttaaa tgcgggaaag acgctgaggc 18360 gttaaaagaa gtttttacca attcaaataa tgtcgccggt aagaaagcga taatggagtt 18420 tgccgggctc tttcgttcag cgctcaacgc caccagtgat tctcctgagg cgaagacgct actgatgaag gtgggggcag agtataccgc gcaaatcata aaagatggcc tgaaagaaaa gtcagctttt gggccatggc tgccagaaac aaagaaagcg gaagcgaagc tggaaaacct ggaaaagcag ctgttagata ttatcaaaaa taacactggc ggtgattaa gtaaattatc gacgaatctt gttatgcagg aggtgatgcc ctatattgcc agctgcattg aacataactt tggctgtacg ttagatccgt taacccgcag caatcttacg caccttgttg acaaagcggc ggcgaaggct gttgaggcgc ttgatatgtg ccaccaaaaa ttaacgcaag agcagggtac cagcgtagga cgggagccc ggcaccttga aatgcaaacg ttgatacccc tgctgctacg taatgttttt gcacaaattc ctgcagataa actgcctgac cctaaaattc cggagcctgc ggctggacca gtacctgatg gtgggaaaa agcagaacct acgggtatta atatcaatat father agcagtaacc atagcgtgga taacagtaag catattaaca atagccgaag ccatgtcgat aatagccagc gccatatcga taatagcaac catgataata gccggaagac gattgataat agccgaacat ttattgataa cagccaacgc aatggcgagt cacaccattc 19200 gactaacagc agcaacgtaa gtcatagtca ttcgcgtgtg gattcgacta cgcatcaaac 19260 ggagacggca cacagcgcca gcacaggggc aattgaccat ggcatcgcgg gtaaaattga 19320 cgtcacggct catgcgacgg cagaggctgt gaccaacgct tcatccgaaa gtaaagatgg 19380 aaaggtggtc acgtcagaaa agggcactac gggtgaaaca acctcttttg atgaagtcga 19440 tggcgtaacc agcaagagca ttatcggcaa gccggtacag gccacggtgc acggcgttga 19500 tgataataaa cagcaaagcc agacggcaga gattgtgaat gtgaagccgc ttgccagtca 19560 actggctggc gtcgagaatg ttaaaaccga taccttacaa tcagacacga cggtaattac 19620 aggtaataaa gccggtacga ccgataatga taatagtcag acagataaga ctggcccatt 19680 ttctggtttg aagtttaagc aaaatagttt cctctcaaca gtaccgagcg ttacgaatat 19740 gcattcaatg catttcgacg cccgtgagac gtttttgggt gtgatacgta aagccctgga 19800 gccagatacc tccaccgt tccctgtacg cagagcgttt gacggcttgc gtgcggaaat 19860 attacccaat ghacgaata agagtgcagc gttgaagcg caatgtagcg atattgaca 19920 gcaccagaa ttgaaagcga aaatggagac tctcaagag gtcattactc atcatccaca 19980 aaaagaaaaa ctggctgaga ttgcactgca gtttgccaga gaggcggggc tgaccaggct 20040 aaaaggggaa actgactatg tgttaagtaa tgtgctggac ggccttatcg gagacggtag 20100 ctggcgagct ggcccggctt acgagtcata cctgaataaa cctggcgtgg atcggtttt 20160 tactaccgtt gatggcttgc acatgcagcg ttattacc gggaaagatg cgatgaat 20220 ggatattgaa gcaagagtca aaaagtgat cacctctgt attgccgttg atgttgatag 20280 tatcaatggt cagacccatc tggttgagga tcttacgct gactcattgg atttaattga 20340 tattgtattt ggtcttagtg aggagtttga cattagttgc aatgaaacg atcttcctga 20400 tatgatgacc tttgcggata tatgccgt tgttaaaaaa agtcttgagt ccagggtgta 20460 gtatttttct ctcgcttgac aacaattgaa tagattaat tgttatgaaa atcatcacac 20520 atgtagtacc cggttcaggt atggctgcaa tatatgatga tattgctgac agttcgcgtt 20580 ttgttattaa aggcaagtta cgtcatgttg aaaatgatcc gaaggagctg ctgatatgtg 20640 ttccgatgcg tagtgaatgg ctattctact ggattaaagg agagaaatat tgcgcccggc 20700 gatgggcgcg aaaaagtatc aagacattgt gtaaccagat taaatttgaa gaagcaatct 20760 tataaaagat ataattcaag aggaaagtaa attgcaagca caccaggata ttatcgctaa 20820 tattggtgag aaattgggtt taccgctcac ttttgacgac aacaatcagt gcttattatt 20880 actcgatagc gatattttta cgtctattga agctaaagat gatatctggt tattgaacgg 20940 tatgattata ccgttatcgc ctgtttgtgg cgattctatc tggcggcaga ttatggtgat 21000 taatggtgaa ctggctgcga ataatgaagg tacgttagcg tatattgatg ccgcagagac 21060 gttgttgctt atacatgcaa ttaccgatct gacaaatact taccatatta tatcgcagct 21120 tgagtcattt gtgaatcagc aggaagcgct caaaaacata ctgcaggaat atgctaaagt 21180 atgaggagag aaaattgaat aatttaacgt tgtcttcgtt ttcaaaagtt ggtgtgtcga 21240 atgatgcccg actttatatt gctaaggaaa atactgataa ggcatatgtt gcgcctgaaa 21300 aattttcgtc aaaagtatta acctggcttg gaaaaatgcc gttatttaaa aacactgaag 21360 tggtgcaaaa acatacggaa aatatcagag tacaggacca aaagatttta cagacatttc 21420 tccatgcact aacggaaaaa tatggggaaa cagcggttaa tgacgcactg ttaatgtccc 21480 gtataaatat gaacaaaccc cttacccaac gtttagcagt gcagatcacg gagtgtgtaa 21540 aagctgctga cgaagggttt ataaacctta ttaagagcaa ggataatgtt ggtgtcagga 21600 atgccgcttt agtcataaaa ggcggcgata caaaagtggc agaaaaaaat aacgatgttg 21660 gagcagaaag taagcaacct ttactcgata tcgcgctaaa gggacttaaa agaacattac 21720 cgcaactgga acaaatggat ggaaatagcc tgcgagaaaa cttccaggag atggcttcag 21780 gtaacggccc gctgcgttca ttaatgacga atttacagaa tttaaataaa attccagagg 21840 ctaaacaact taatgattat gttacaacgt taacaaatat tcaggtcggt gtagcccggt 21900 tctcacaatg gggaacctgt ggaggagagg ttgagaggtg ggttgataaa gcctctactc 21960 atgaattaac ccaggctgta aaaaaaattc acgttattgc taaagactg aaaaatgtta 22020 cagctgaact tgagaaaata gaagccggcg cgccaatgcc acagacgatg agcggaccga 22080 cactgggcct ggcacgattt gcggtcagca gcattccaat taatcagcaa acccaggtga 22140 aactgagcga tggaatgcct gtgccagtga atacgttaac tttgacggt aagcctgtgg 22200 cattagccgg ttcgtaccca aaaaatacgc cggatgcgct ggaagcgcat atgaaaatgc 22260 ttcttgaaaa agaatgctcg tgctgtgg tgttaacgtc ggagatcag atgcaggcaa 22320 aacaattacc accctattc agagggagct acaccttgg cgaggtgcat accacagcc 22380 aaaaagtgag ctcagccagt cagggagag cgatagacca atacaatg caactgtcct 22440 gcggggaaaa gcggtataca atcccggtat tgcatgtgaa aaattggcca gatcaccagc 22500 cgttaccgtc tacggatcag ttagaatacc tggcggatag ggtgaaaaat agtaaccaaa 22560 atggcgcgcc ggggcgtagt tcatcagata agcatttacc gatgattcat tgtctgggcg gagtgggag aaccggacg atggcggcgg cccttgtact tag cctcatagta atctggagca ggtacgtgca gatttccggg atctcacgga caatagaatg ctggaagacg cctcacagtt tgtacaacta aaagcaatgc aagcccagtt gcttatgacg acggcaagct gataaaaca tagcttactt tagcttactt ctgaaagtaa gctatttctg father tgtttgccgc gatagaaagt cttttgtttt tctcttctgc tgacattcct tttaattttc tgtaattctc ccaaattttt ttagcataac gtttccttat atccgatcgt ttcggcgacg tcccggcatt fathercgcca acggcctccc father gtagatttc atcatatctg ataaatgga ggcgcccaca atgacagaaa tgcaggggtc ctgtaacaac tgtttttcac taatccccat ttttttcagc cttttcatat ggaagctgtt aatttgcatc aggccaagat cggttgaacc atctcggtta tgaccaatgg cgccaggttt catcgccgat tcctgctggg cgatagcgta aagtagttcg gattcaatt tgaacatttt ttcagcctga agccagcaat cagcccatgc cgtatttatg ctaagcaacc agattacgat gataaaaaaa taatgcatat 23340 ctcctctctc agattttacc gtaatttaat caagcgggga tcctgtttcc atcttttgaa 23400 ccaaatattg tcttcgtttt taaatttatt ccacattttc tcggcaatag cttcgccgtg 23460 ggcaaccagc actaacggta ataatcccgg attattatct atacgctgtt cactttccag 23520 aaattctctt atcgtcggta aggcacgctc actattctga caatattcag cgtaaagaag 23580 attaacagca ataagtcctg ttatttcctg cgtggatatt tctttagtta attttcgtgc 23640 cagttcatgt ttacctttaa gcgaaagaaa cataacctgc atacttaata atattggatt 23700 atcctgcagg tgttgtgagc gtaattcatc gcctaaacgt atagcatcat caataccggt 23760 atgataatag gtaatccaca gcttagtgat ccctgcggct gcgcgcgttg ggtccaattt 23820 taaacactcg ttaatcgttt gtaaggcctc ctccaactga ccagccatga aaagattcca 23880 gccataataa tatttaatat ctgcagaaat gggcgaaagt aagttagctt gtttgaatag 23940 caaactcccg acgatgtatt ctgagtgaat cgtattaatt agccccagta atcctaaagc 24000 ttgtggatta ttgtggtcca gctctgtcgc cttaatcgca tgttctttag ctttgatcat 24060 agcgttttgt ttatcaaaaa tccccatttg cgccatgctg aggtagcatt ctgccagcgc 24120 acagtaaggc gcaatgctgt ttggcgacat gttaacgcat tgagtcagca atttaagcgc 24180 ttgctgtaag ctataggggg tgtattgatt taactcatgc ttaccgcgta agtaaaccat 24240 agtactgtca atcgaattta gctcgctaat ctgctttgtg tcccagcgaa gtccgggaat 24300 acatctgagc aaaagattcg caattttgtt ttgcaagaga gaagcgggtt ggtgttctat 24360 caacttaatg ctttcctggt gcagcaggtg ataaccttta acccgaacta tctcaatctg 24420 tacaatatta tcattaccat cgggtatcat ctgcccggag atataataat cggggcgtaa 24480 ttgatccatg agctcaagaa tatccttaac actgcggcag ttcttcgtaa tggtcaccgg 24540 cagcacgctc aggccaaagg gcgcatactg cgataatccc ttcacgatag agtaatgcag 24600 actctcggat tgaacctgat cctgcatctg aaaaggaagt atcgccaatg tatgagtcgt 24660 aggttgcggc gctggcggag acaccactac gaccggacga ttaaaccgat agccctgtcc 24720 gtacagtgtt tcaatgtaac gatgctcttt atcttccgac agaatacgtc gtaaggcata 24780 aatacagcgg gtaagagatt cttcgttaac ttccgcgtcg ccccatacct ggtccagtaa 24840 ggtgttttta ctcacaatct cgccggcggc ttcgagcagg atgaccagaa cggcatattc 24900 ttttggcgga atattgactt tcttttctga gcgtagcagg gagccgtcca tgttgagtat 24960 gaaatcatca aagacgaatt ttttattcga tacaggaaca ggattaaaat gtggcatgat 25020 aatagtgtat tctcttacag ggtgaaaagt tataataact aatacaagtc gttaggatag 25080 aatctactgt ttcagtaatt ctgctgttag tactaacggt caggttgagc ttttattatg 25140 agcttaatgt catattctac gggctcgggt ttatatgttt ttaacatttg attatttagc 25200 ttatgcctgt gctttaaata actgacatgt tggtaataat attgttataa ctaactgtga 25260 ttatcgaatg gacgctgccg tatatcctgg tgcagagaga gtcagcgccg ggcatctaaa 25320 tatttacata tttaacttgg cctcattgca atagagatta gttttgtagc tatcttactg 25380 cattttttg gggtgtaaat gctgcttatt atatcttcat ggttaatggt ctgaataatta 25440 caccatcaga accatttttc tcgaagattt ccactaaatg accattcgtt gaaagtttaa 25500 gagtgtaatt tgtcttaggt tccaaaatat aaatgaaaga ctgtttttaa tggtgcgctt 25560 atttttatat ttttttgcaa aataatgata tcgtcttttc tttactccct tcagttatag 25620 cgtcaaaaag gcaggagggt tatgagcagt atcatcctca ggctggctcc aggcattata 25680 tcgatatcag gaggaagaga ggaggtatgc ctggcagaaa gctaacaagc gtgactgttt 25740 cggtataaac cgttcaggca aatagttctc agagggaacg gatgatgtat aaatatgaat 25800 aaaatgccgg ccttaatcca cagggttaaa gccggaagga gatagtgtca tctggacgaa 25860 ttcttacgat gtgaataagg caatatcgtt ttaccagtca tcatagcgat gacaagttca 25920 caaaaatcgt gttatataat cagccttact ttaaattacc atagatatca gtataagctg 25980 tcttccggac agaacaatga tattgaatag cctcccatcc tgatagagcg tgttaatgcg 26040 cagtctgaat tttaaattat tacaatttta ataaaaatct ttacttaagt gacagataca 26100 aaaaatgtta atggttcgcc attttatga atgtcgatgg cgtagtttta aaatattttt 26160 tgaaacattg aatgaagtag gacgtgctat cataaccaca ttttaatgct acagcattaa 26220 cattatggtt gcctatgcgt aaaagttttg ctgcctgatt cattcttgcc gataagtaga 26280 tgtcgctaaa gctggtacct tcttctgcaa gtttccgttt gagcgttgac gtactcataa 26340 atatatgatc ggcaacatcc gtaagcttcc actgtctact gggtgacgaa gatataatgt 26400 tgtaaacgcg ctcctttaac gttatctgag ccgagctaag gatgatctca taactctccg 26460 ggagtcggag aaaagcagta agcaggaaca gcagaaaata aggaattaac tcttcatttt 26520 caatacaact tttgttgtta cagttactgc aacctttgct tgtatccttc gtacaggaga 26580 acgccgttt cagatgttca aatacctctc ttctggcagg aaagtcaggc gtatagaaga 26640 tcttctgcgc tttctctgtg ggtaccgcca ttttggtttg ctgctcgttt gggataagtg 26700 catagagagc gccaagtcgt tgcgtcggta tctcgagtat atcgaaatcc atgtggccat 26760 tgacctcttc aagcgtcacg ttaactacct gctctttttt gagcaccaac atcccaggtt 26820 cgtcacagta aatcgtatcg tgattattgc taatcgtcag tttaccgctc cgaaagcaaa 26880 ctaaagtgaa actgcttaca taaagatttt tgatggtaac ctgctgagtc tgacttttaa 26940 tttgctgccg ggtatttgtc aaaagtgatt ttaatttctg taagttatct gcggcaggac 27000 gctgatgact attacttaca aaggttacat tttccatatt atccctttgt tgatgttatt 27060 ttaatgttcc ttactggtat cctactgaaa aaatctgagt tgtaaatgct ctttattagc 27120 gtgtgttggc aatggtctga ttgttacacc aaaagaaccc aaatttgggt aatttatcta 27180 cagtagttta agccccaatg gggatgatgg ttcttttaat atgtgttgag acgcattata 27240 cagaataaat tgattttatt tctcactttt cattctattt tcatcaggaa tccctgtgtc 27300 ctgtgcggta atctgctgct atcgagaacg acagacatcg ctaacagtat atatggaaac 27360 atcaaaagag aagacgataa caagcccagg gccatacata gttcgattac ttaacagctc 27420 actgaacggc tgtgagtttc cattgctgac aggccgaaca ctctttgtgg taggtcagag 27480 tgatgcgctc actgcttcag gtcaactccc tgatatacct gccgatagct tttttatccc 27540 gctgggaccat ggcggagataa attttgaaat ccaggtggat acggatgcga ccgaaattat 27600 actccatgag ctgaaagaag gaaattctga atctcgttcg gtgcaattaa atacgccaat 27660 acaggtcggt gaattgctta tcctgattcg cccggaaagc gagccgtggg tgcccgagca 27720 gcctgagaag ttagaaacgt ctgcaaaaaa gaacgagccg cgttttaaaaa acggaattgt 27780 agcagcactg gccgggtttt ttatattggg aattgggact gtggggacgt tatggatact 27840 taactcgccg cagcggcagg ccgcagagct cgattcgtta ttggggcagg agaagggcg 27900 ttttcaggtg ttgccaggcc gggacaaaat gctctatgtc gctgcgcaaa atgaaagaga 27960 tacgttgtgg gctcgtcagg ttttagcgag gggcgattat gataaaaatg cgcgagtgat 28020 taacgaaaac gaaaaata agcgtatctc tatctggctg gatacctatt atccgcagct 28080 ggcttattat cggattcatt tcgatgagcc gcgtaaaccc gttttctggc taagccgcca 28140 gcgaaacacg atgagcaaga aagagctcga gttgttaagt caaaagctga gagcgctaat 28200 gccttacgcg gattcggtta acatcacgtt gatggacgat gttaccgcag caggccaggc 28260 ggaagcgggg ctaaaacagc aggcgttacc ttattcccgc aggaatcata aggggggcgt 28320 aacgtttgtt attcaggggg cgctcgatga tgtagaaata ctcagagccc gtcaatttgt 28380 cgatagctat taccgcacat ggggaggacg ctatgtgcag tttgcgatcg attaaaaga 28440 tgactggctc aaggggcgct catttcagta cggggcggaa ggtatatca aaatgagccc 28500 aggccattgg tatttcccaa gcccacttta attaacgta aataaggaag tcattatggc 28560 aacaccttgg tcaggctatc tggatgacgt ctcagcaaaa tttgatacgg gcgttgataa 28620 tctacaaacg caggtaacag aggcgctgga taaattagca gcaaaaccct ccgatccggc 28680 gctactggcg gcgtatcaga gtaagctctc ggaatataac ttgtaccgta acgcgcaatc 28740 gaacacggta aaagtcttta aggatattga tgctgccatt attcagaact tccgttaatc 28800 agttataagg tggattatgt cgattgcaac tattgtccct gagaatgccg ttatagggca 28860 ggcggtcaat atcaggtcta tggaaacgga cattgtctcg ctggatgacc ggctactcca 28920 ggctttttct ggttcggcga ttgccacggc tgtggataaa cagacgatta ccaacaggat 28980 tgaggaccct aatctggtga cggatcctaa agagctggct atttcgcaag agatgatttc 29040 agattataac ctgtatgttt ctatggtcag tacccttact cgtaaaggag tcggggctgt 29100 tgaaacgcta ttacgctcat gattcgtcga tatctatata cttttctgct ggtaatgacc 29160 cttgccggct gtaaggataa ggatctttta aaaggactgg accaggaaca ggctaatgag 29220 gtcattgccg ttctgcaaat gcacaatata gaggcgaata aaattgatag cggaaaattg 29280 ggctatagca ttaccgttgc tgagcctgat tttaccgctg cggtgtactg gattaaaact 29340 tatcagcttc ctccccggcc acgggtggaa atagcgcaga tgttcccggc ggattcgctg 29400 gtatcgtctc cgcgagctga aaaggccagg ttatattcgg ctattgaaca gcgactggaa 29460 cagtcattac agacgatgga gggcgtgctc tccgccaggg tccatattag ttatgatatt 29520 gatgctggtg aaaatggccg cccgccaaaa cctgttcatc tgtcggcatt agccgtatat 29580 gaacgaggtt cgccgcttgc gcatcagatc agcgatatca agcgtttctt aaagaatagt 29640 tttgccgatg tggattatga caacatttct gttgtgttgt cagaacgttc tgatgcccaa 29700 ttacaggctc ccggcacacc agtaaaacgt aattcttttg caaccagttg gattgttttg 29760 attattttgt tatccgtgat gtcagcaggc tttggcgtct ggtattacaa aaaccattat 29820 gccgcaata agaaaggcat aacggctgat gataaggcga aatcgtcaaa tgaataggca 29880 gccattaccc attatctggc aaagaatcat ttttgatccg ttatcgtata tccatcctca 29940 gcggttgcag atagcgccgg aaatgattgt cagacccgcc gccagggcgg cggcaaatga 30000 gttaatactg gcggcatggc ggcttaagaa cggagaaaag gagtgtattc aaaactcact 30060 gacgcagctg tggctgcgtc agtggcgccg actgccgcaa gtagcgtatt tactcggttg 30120 cataaactg agagccgatc tggcaaggca gggagccttg cttggcctgc cggattgggc 30180 gcaagcattt ttggcaatgc atcagggaac aagtttatct gtctgcaata aggcgccgaa 30240 tcaccggttt ttacttagcg tcgggtatgc acagttaaat gccctaaatg aatttttacc 30300 tgaatcttta gcacagcgtt ttcctttgct ttttcctcca tttattgagg aggcattgaa 30360 gcaggatgct gtagaaatgt caattttgct actggcctta caatatgctc aaaaatatcc 30420 caataccgtc cccgctttcg cctgttgagg ggatactgat aaagcgtaaa acgctggaac 30480 gttattctc tattgaaaga ttagaacagc aggcgcatca acgggctaag cgtattttgc 30540 gagaggcgga agaagaggca aagaccttgc ggatgtacgc ctatcaggaa ggttacgagc 30600 agggaatgat agacgcgtta cagcaggtcg ccgcttatct tactgataat cagacaatgg 30660 cctggaagtg gatggaaaaa atacagattt atgcccgcga gttattttca gctgcggtcg 30720 accatcccga aacgctttta accgtcctgg atgagtggct aagggatttc gataagcctg 30780 aggggcaact ttttttaaca ctgccagtta atgcgaaaaa agatcaccaa aaactgatgg 30840 tgttgcttat ggagaactgg ccaggcactt ttaatcttaa atatcaccag gaacagcgct 30900 ttatcatgag ttgcggcgat cagatcgcag aattttcacc tgaacaattt gttgaaacgg 30960 footggcgt cattaagcat catcttgatg aacttccaca agactgccgg acaatttctg 31020 ataacgccat taacgcactt attgatgaat ggagacaaa aacgcaagcg gaggttataa 31080 ggtgataccg ggtacgattc cgacttctta tttggtcccg acagcagata ctgaagcgac 31140 gggtgtggtt tcgttgagtg ccagggccgc tatgctgaat aatatggata gcgcgccttt 31200 aagcaatggc ggcgacgttg atctttatga tgcattctac caacgacttt tggcgcttcc 31260 tgagtcagcc tcttctgaaa cgctaaaga tagcatttat caagagatga atgccttcaa 31320 agatcctaac agtggtgatt cggcttttgt ttcttttgag cagcaaacgg ctatgctaca 31380 aaacatgtta gcaaaagttg aaccgggcac ccatttat gaggctttaa acgggttatt 31440 agttggttca atgaacgcac agtcacaaat gacctcctgg atgcaggaga ttatttattc 31500 gggcgggggaa aaaagagg caattgactg gtgaaccccc attgctggcg atttgtgata 31560 aggtatggtg tatatatcag caaataatgc agaaaatgca ccatgccagt gtgtctatct 31620 tattgatgta tattgatt tagatgtct gccagccgtt tattctgaa atgattatat 31680 taatattgta agctttcatt acaaaattgt gcataaagtt tgtaagggta tctttttgtg 31740 acgaatgaa cgcgcgttgg atgtatta ttggctataa taaaaaaa atcggattta 31800 aatcatctct gtgagtttta attatttgtt tataagaata taacgatttt gagttcctta 31860 tagcacacag gataaaatat ggtattgcct tcaatgaata atcagttga ggccattagc 31920 aaatcacc ttcaacagcc gacaaattt ccattaataa atggattagc tgacgtaaga 31980 gactattatg tcgcaactg cttattgttt aaacttaata aaggcagttt gcgaattgaa 32040 aacgaatttg gggagttcat cgaacgatct gcgccgtgtt tattttatt ggaaaagat 32100 cieacaatca cgcttagtat gagcgaaata gaagggcata ttgattttc ttcactggaa 32160 gtttcctatg acttaatgca aaattctac aaagtttttt acagtacag aaactataat 32220 gatcgagagc tatcattaaa aacgaaacca aagtattttt ttcatgcgga cttgttgcca 32280 gggatgagtg atacttttga ctctattttg catggtgtgg catgtccacg ggtttgtagt 32340 aatgtgagta ttgatgatca tgattattca tatttctcat tgatgtatct tatatcggca 32400 tttgtacgta agcccggtgg gtttgatttc cttgagcgag caataaaaat tacgacaaaa 32460 gagaaagttt ataacattat tatcagcgat ctcacccgca aatggtcaca ggctgaggtg 32520 gcaggaaagc tgtttatgag cgtatcaagt ctgaagcgaa aactggccgc tgaagaggtg 32580 agttttagta aaatatacct ggatgctcgt atgaatcagg ctataaaatt attacgtatg 32640 ggggctggaa atatttcaca agtcgcgacg atgtgtggct atgatacgcc ttcttatttt 32700 atcgctattt ttaaacggca ttttaagatt acaccgctta gctttatgcg tacaatgaac 32760 cattgatttg tgaaattatt ttaaacgtta ctatctgttt gcgttacaga taccaaatat 32820 ttttttaatc gtagtgtata tttcagtatc gctacaccag tgatatgtag ttaatgaaag 32880 ggaatcgaaa gcgttcattg tcagacatac atggccacca ggcgccgggc aatcgttgta 32940 ctctaagaga ttacagtaaa gtaaaacagc aaatttaatc attatcagat ttgtaaaagg 33000 aattcatttc ggtaacgaca ttgcgcatgt aataacgtat ctggtcttat tacctttgta 33060 gtaagaaggc gttatctact tgcgcagtag tcgcccagga agaaaaatca ttaccgaagc 33120 tgtggtcagt tttcaaaaaa ggataaataa atgagaaatg taattatata cggtattaac 33180 tggactaatt gttatgccct acagtctatt tttaagcaaa aatatccgga aaagtgcgtt 33240 aaaacgtgta actcattaac tgcacttttg cattccctat ccgatatgcc ggacgccggg 33300 ctgattttag ctttgaaccc gcatgagcat gtttatcttt ttcatgcgtt gctgacgcgg 33360 ttgcagaacc gtaaagtttt ggtagtggca gatcgcttgt attatatcga tcgctgtgtg 33420 ctgcaatatt ttggcgttat ggattatgtt ttaaaagacg aactatcctg cgctatccgc 33480 tcggagcgag agaagctccg tcttccggaa gcctggctgc gtttctgcca caggccacaa 33540 aagaaaaccg tggctgctac gtatgcttt aatgccggcg agacgccgga agaggtattg 33600 tttaatatca atcaatacgc ctggtggaat ctacctcccg gtgtcacgca ggcgaaatat 33660 gcgctgctga tattattatc ctccggacat ccggcaattg aactggcgaa aaaatttggt 33720 ctgggaacaa aaaccgtcag tattatcgt aagaaggtta tgtatcgttt ggggatggac 33780 tcttcaccgc tatcgctgtt ccggggactg aaactagacg cgcatttaca gcgcactgct 33840 tttgcacata accctgctgt acctgacgat aattgcgcgt taccgattgc ggtaggaatg 33900 attaaaata tttttatca tttatgacca tgtgttgagc ttttattata aaaaagattt 33960 tttgagtagt aattcttata tataatcatc cggaggtggt tggtagcctg gctcaatcat 34020 tgaggcatat ttttgcaggc aatatttga atctgaaaag ttaaagatga tattttcggt 34080 gcaggagcta tcatgtggag ggaaaagtat gctaagtcct acgactcgta atatgggggc 34140 gagttatcg cctcagcctg acgtcagcgg ggagctaaac accgaagcat tgacctgtat 34200 tgttgagcgt ctggaaagtg aaattataga tggcagctgg attcatatca gttacgagga 34260 aaccgatctc gaaatgatgc cttttcttgt tgcacaggcc aataagaagt atccagagtt 34320 aaatcttaaa tttgttatgt cagtccatga gcttgtttcc tcttaaagg agaccagaat 34380 ggaaggcgtt gatctgccc gatttctcgt aaatatggga agttcaggta tccatatttc 34440 agtcgtcgat tttagagtta tggacggaaa gatacggtg attttgttcg aaccagcagc 34500 gtgtagcgct tttggacctg cactggcgtt gaggaccaaa gcagcttg aacgtgaaca 34560 actgcctgat tgttatttg ctatgtcga gctggacatt caacgaagct cttctgaatg 34620 cggtattttt agcctggcgc tcgccaaaaa acttcagctt gatttatga acttagtaaa 34680 aattcatgaa gataatattt gtgaacgtct gtgtggtgaa gaaccttttc tcccgtccga 34740 taaagcagac cgctatctgc cggtgagtttt ttcaacat actcaggcg cacaacgatt 34800 aaatgaatat gtggaggcca atccggcggc gggaagcagt atagtaaca aaagaatga 34860 aacgctttat gagcgattcg atacaatgc cgttatgcta aacgataaa aactctctat 34920 atccgctcat aaaaaagga tagctgaata tagtcttta cttaaaccgt atgaaatta 34980 taaggttgcc tgccagagga aatccttcca gc 35012 <210> 2 <211> 25440 <212> DNA <213> Unknown <220> <223> Salmonella pathogenicity island-2 <400> 2 ttttgtcgat gatgagcaat actgcgtggc gtaaggctca tcaaaatatg accaatgctt 60 aataccatcg gacgcccctg gttaatactc tattaacctc attcttcggg cacagttaag 120 taactctgtc actttatgaa cctgtagctt tctcatcata ttcatccggt gtgtttcgac 180 ggtttttata ctgatatgta gcttttcgct gatcccatga ttggtatacc cctcgtcaat 240 aagtttaaga acctgacgct cgcgcaaagt aagcagttga tgattggtcg tgtcagcgtt 300 taattcagcc aggatagctt cccgattcaa cgttgggtca atgtaacgct tgtttactgc 360 tactgtttgc aatgccgcta acagaacttg ctgactactg ctttttaaaa catagccatt 420 agcacctgcg gctaaagttt taatggtcat atactcttgt tggtatgctg tgtaaaccag 480 aatattcatt gctggccaac gctgatgtaa ttgaggaatg atatccaggc cattgatgcc 540 aggtagacta agatcaagga taagtatgtc aggctcgtat gcacaacagg cattataaac 600 ctcaagacca tttttaacat gctctacaat tttaaaatga ggccagggta ataaggcatt 660 cataatgccg ttaatgatga tttcatgatc gtctactaat aagatcttat attctttcat 720 tttgctgccc tcgcgaaaat taagataata ttaagtaatg gtgtagtttt tgaagatcat 780 acgtattttc tggcgtaagt cggttagttc ctccagcgcg atgattttcc ccatttttac 840 gcgattctca atgtctatga catagcatac caattcagtc tgccctattt gacctaaaca 900 gcctttaat gtgtgaatta actgatcgat tttttctcca gccgatacgg cattttcaat 960 atcagccagc aagaggtcca gtgattggaa aatcttgcta ttaatgacca tatcatctgt 1020 cgccagtagc gctgagcagc gactcggatc ctgctcctgt agctctatat ttcgtaaaag 1080 ttggtattct gcggcaatac tgatgtagcg agctaaggta gccaatgtca ctggttttgt 1140 aatgtaatga tgaatcccat tttttttaca acgatgaata tcttctgtcg ctacgctagc 1200 ggatagtgcc acaaacatgc agtcaggatc taaattattc ggctcatcat gccataatcg 1260 tacacattca atacatcta tttctggcat tctaatgtca atcagtacta aatcgaatcg 1320 ctgctgttgt gataaagtca gagcctcgtt actactggcg gcaatagtga cgtgttggcc 1380 caggctgaca agcattttgc cgatgatatc ccgattaata tcggcatcat caaccaaaag 1440 aatctgcaac tgccagggtg gtaacaaatt atttattact ggcatatttg gtgtacacaa 1500 tattaactgt tgcgccaggt cgtagagttt tccggagaaa tacaaaagct ctgcgttgag 1560 aagcgcattt tgctggtggg gtggttcacc gcgtattccc cagcaagcca gttgccgatg 1620 caggcagaac ggcgctgaca gcgtcccttt aattggttga ggcggctggt attcttgtaa 1680 gggtaatact agcgagacac aggttccaac cccggggaca ctttttagtg tcagattacc 1740 gcccatcatt ttagccaggc ttgacgcaat agtcagtcca attcctgtac cttgcgaatt 1800 tgtgtctgct tgataaaaag cagtaaagat ttgagactgc tgctgtattt caatcccttt 1860 accgctatcg ctaaccagaa atattaattg ttcctcatga cgcttgaccg tcagacgtat 1920 ccctccggtt tcggtaaatt ttaccgcgtt cccgagtaaa ttaaccaaaa tttgccgtaa 1980 acggatactg tcggtatgaa aatagagagg gacatgttga ccgacaaaag tacgtaatga 2040 cagttttttg ctttgcgctg gccctggat ggtttgcatt gcctggtcca gtaacggcag 2100 taacgctgtt tcttccatat gtaatgtgaa atgaccagac tcgatgcgtg aaaaatccag 2160 cagattatta atatagcta acaaagacag tgtacaattt ctggcggtat cagctaatcc 2220 ttgttgctct atgtttaaag gggtggtttg tataattca attgcaccga gtacgccatt 2280 catcggagta cgtaactcat gacttattac cgtaagatga atgcttttac gtttgttagc 2340 tcgctcagcg cgttttttg cttcatttag cgcctgggtg cgctctgcga ctttgttttc 2400 cagattgtcg tattggactt gtagagtatc aagcagttgg ttaaaagcac cggcaatact 2460 atctaattca tccagtcgtt gtgctggtaa acgtgtgctc agcggtgcag ttgcggtttt 2520 attaatgaca tcgacaaaac gccataacgg tttggccagt gagcgatgta gtaaccagca 2580 aaaagccgac gtcatcaaca ccaatgctgt taatgtaaag gggatttgtt gaaggataat 2640 ttttaagatg cgattatgta gattaccgta tgggtacagc gtaaccagac tccatccggg 2700 gccatgcaag gttgtgcgta atatcagaaa tccgggaatt tgctgccatc catcatgcag 2760 cgttacattt tctaactgtg tacgtatttt ttgcgggatg tatgaaaacg gcaataagtg 2820 gttgttttga tccagccata ctcgaatact atcatctaat ggcaggtggc tcttagtaat 2880 gagatcggga agtttaccg tcacccaa aaatacgcct tgctgatcgg caccgcaac 2940 ggaagcgtgc catcctttgc cgtttagta ttctggttca ctccagtaaa acccggcatg 3000 ggttgggtat aaggaaagc ttttcgcgt taaagctgt agagttgaat aatctgaagg 3060 gttatcagta gataacg aaatctcatt tttgatta agataaaac tatcgcgacg 3120 aaagctattt tcatcgatat cagaggactg cagaaagaga cggtgcttct ctccgtttag 3180 cgtcggcgtg caatttgaag gaccgacaga tegtgccgg ctcacctcag ggaaaataatc 3240 gttatgatga atctcagtcg ctaatgagca ttgatacatt aaatttttag cgtcacgttc 3300 agcttcttca aaccgttgat tgcttagtac atattcatc tcggatagaa cggataaatc 3360 cttatata tgctgccgtt tctgaaccat tgatatata gctgcggtaa gcacagatag 3420 cagccaaata attattgttg ttaaaaa taaaaaagtt agcctgatta ctaaagatgt 3480 ttgcagcgta ttcttgagat tgagcaaatt cataatgctt ccctccagtt gcctgttgca 3540 aaatctttgg cacttgatca ctatcgcagt acatatagtt tcatcagaag attaatcgat 3600 ggtgttatca ttaggaagat aaatttcttc atatataacc cagtcgatga ctacaattac 3660 tttttaataa gatggcgatg taaaaacatc gtaacagttt atttaataaa taatttttca 3720 aattgtaagt tttatgtca atgctgaaaa tgtaattgtg aatttatcgg aaaatccgaa 3780 tgatagaatc gcctgtgaca aggtatatgt agacagcatc ctgatattgt acaagaagag 3840 tatagtcgaa ataaatgtga atcaggcttt ttacggatgt ggttgtgagc gaatttgata 3900 gaaactccca tttatgtctg aggagggatt catgctggca gttttaaaag gcattccatt 3960 aattcaggat atcagggccg aaggtaatag ccgatcctgg ataatgacta ttgatgggca 4020 tcctgccaga ggagaaattt tctcagaagc attttctatt tctttgttct taaatgacct 4080 ggaaagctta cctaagcctt gtcttgccta tgtgacacta ctgcttgcag cacacccgga 4140 cgtccatgat tatgctatac agctcacagc ggatggggga tggttaaacg gttattatac 4200 cacaagtagt agctctgagc ttattgctat tgagatagaa aaacacctgg ctttaacttg 4260 cattttaaaa aatgtaatac gcaatcacca taaactttat tcgggtgggg tataaaatgg 4320 tagtaaataa acgtttaatc ttaattttac tatttatact caatacagca aagagtgatg 4380 agttatcatg gaaaggtaat gacttcaccc tttatgccag acaaatgcca ttagcagagg 4440 ttttacatct gctctcagag aactatgata cggctattac tattagccca ttgataacag 4500 ctacatttag tggaaaaatt ccgcctggac caccggtcga tattttgaat aacctggcag 4560 cacaatatga tttgcttacc tggtttgatg gcagcatgtt atatgtatat cctgcatcgt 4620 tattaaaaca tcaggttatc actttcaata tttatctac tggacggttc attcattact 4680 tacgcagcca gaatatcctt tcatcaccgg gatgcgaggt taaagaaatt accggtacca 4740 aagctgtgga ggtgagcggt gttcccagct gcctgactcg tattagtcaa ttagcttcag 4800 tgctggataa tgcgttaatc aaacgaaaag acagtgcggt gagtgtaagt atatacacgc 4860 ttaagtatgc cactgcgatg gatacccagt accaatatcg cgatcagtcc gtcgtggttc 4920 caggggtcgt tagtgtattg cgtgagatga gtaaaaccag cgtcccgacg tcatcgacga 4980 acaatggttc acccgctaca caggcattgc ccatgtttgc tgccgaccca cgccagaatg 5040 cagtgatcgt tcgtgattat gcggccaata tggccgggta tcggaaactc atcacagaat 5100 tagatcaacg ccagcagatg atagagattt cggtgaaaat tatcgatgtt aatgctggag 5160 atattaacca gttaggcatc gactggggaa cggcagtgtc gctgggtggc aagaaaattg 5220 cgttcaatac aggtttgaat gacggtggtg ctagtggtttt ttcaacggta atcagcgata 5280 cctcaaactt tatggtgcgt ctgaatgccc tggaaaaaag ctctcaggct tatgtacttt 5340 cccagccatc tgtggtgact ttaaataata tccaggctgt gctggataaa aatattactt 5400 tctataccaa actgcaggga gaaaaagtgg ctaaacttga atccatcact acgggttctt 5460 tgttacgcgt tacacctcgc ttgttaaatg acaatggcac gcaaaaaata atgcttaatc 5520 ttaatattca ggatggacaa caaagtgata cgcaaagcga aacagacccg ctgcccgaag 5580 tgcaaaattc tgaaattgct tcgcaagcca cattattggc cgggcaaagt ctattgctgg 5640 gagggtttaa acaaggtaaa caaatccact cgcaaaacaa aatcccttta ttgggcgata 5700 ttcctgttgt aggtcatttg tttcgcaatg atacgactca agtacatagt gtaatcaggc 5760 tttttttgat taaagcctca gtagtaaata atggcatatc tcatggttaa tccaaagagt 5820 tcctggaaaa tacgtttttt aggtcacgtt ttacaaggcc gggaagtatg gctgaatgaa 5880 ggtaacctgt cactggggga gaagggatgc gatattgta ttccgctggc tataaatgaa 5940 aaaattattc tgagagaaca ggcagatagt ttattgttg atgccgggaa agccagagtt 6000 agagttaatg gccgcagatt taatccaaat aagccgctac catccagtgg ggttttgcag 6060 gttgcgggag tggctatcgc gtttggtaaa caggattgtg aacttgctga ttatcaaata 6120 cccgtttcca gatcagggta ctggtggttg gctggcgtat tcttgatttt catcggtgga 6180 atgggtgtcc tgttaagtat tagtggtcag cctgaaacgg taaatgactt acctttgcgg 6240 gttaagtttt tattagacaa aagcaatatt cattatgtgc gggcgcaatg gaaagaagat 6300 ggcagcctgc agttgtccgg ttattgctcg tcaagcgaac agatgcaaaa ggtgagagcg 6360 actctcgaat catggggggt catgtatcgg gatggtgtaa tctgtgatga cttattggta 6420 cgagaagtgc aggatgtttt gataaaaatg ggttacccgc atgctgaagt atccagcgaa 6480 gggccgggga gcgtgttaat tcatgatgat atacaaatgg atcagcaatg gcgcaaggtt 6540 caaccattac ttgcagatat tcccgggtta ttgcactggc agattagtca ctctcatcag 6600 tctcagggggg atgatattat ttctgcgata atagagaacg gttagtggg gcttgtcaat 6660 gttacgccaa tgcggcgctc ttttgttatc agtggtgtac tggatgaatc tcatcaacgc 6720 atttgcaag aaacgttagc agcattaaag aaaaaggatc ccgctctttc tttaatttat 6780 caggatattg cgccttccca tgatgaaagc aagtatctgc ctgcgccagt ggctggcttt 6840 gtacagagtc gccatggtaa ttacttatta ctgacgaata aagagcgttt acgtgtaggg 6900 gcattgttac ccaatggggg agaaattgtc catctgagtg ccgatgtggt aacgattaaa 6960 cattatgata ctttgattaa ctatccatta gattttaagt gagtggaaaa tgacaacttt 7020 gacccggtta gaagatttgc tgcttcattc gcgtgaagag gccaaaggca taattttaca 7080 attaagggct gcccggaaac agttagaaga gaacaacggc aagttacagg atccgcagca 7140 atatcagcaa aacaccttat tgcttgaagc gatcgagcag gccgaaata tcatcaacat 7200 tatttattat cgttaccata acagcgcact tgtagtgagt gagcaagagt aaagtaaaaa 7260 tatcttagag cctatcccac caggcgttaa ttggcgcagc cagtttggac acggatagcg 7320 cgcaaaaacc gcagcgtaca cgtagtacgt gaggtttgac tcgctacgct cgcccttcgg 7380 gccgccgcta gcggcgttca aaacgctaac gcgttttggc gagcactgcc caggttcaaa 7440 atggcaagta aaatagccct aatgggatag gctcttagtt agcacgttaa ttatctatcg 7500 7560 gagcaaagtt acatgcagct aaatcactgt cttaaaaaat ttcaccaaat ccgggctaag 7620 gtgagtcaac agcttgctga aagggcagag agccccaaaaa atagcagaga gacagagagt 7680 attcttcata acctatttcc acaaggcgtt gccggggtta accaggaggc cgagaaggat 7740 ttaaagaaaa tagtaagttt gtttaacaa cttgaagtac gactgaaaca acttaatgct 7800 caagccccgg tggagatacc gtcaggaaaa acaaaaaggt aaagcataat gtcttcagga aacatcttat ggggaagtca aaaccctatt gtgtttaaaa atagcttcgg cgtcagcaac gctgataccg ggagccagga tgacttatcc cagcaaaatc cgtttgccga agggtatggt gttttgctta ttctccttat ggttattcag gctatcgcaa ataataatt tattgaagtc cagaagaacg ctgaacgtgc cagaaatacc caggaaaagt caaatgagat ggatgaggtg attgctaaag cagccaaagg ggatgctaaa accaaagagg aggtgcctga ggatgtaatt aaatacatgc gtgataatgg tattctcatc gatggtatga ccattgatga ttatatggct aaatatggcg atcatgggaa gctggataaa ggtggcctac aggcgatcaa agcggctttg gataatgacg ccaaccgga taccgatctt atgagtcagg ggcagataac aattcaaaaa atgtctcagg agcttaacgc tgtccttacc caactgacag ggcttatcag tagtggggg gaaatttcca gtatgatagc gcagaaaacg tactcatga aaaagacccg accctacaac aggcacatga cacgatgcgg tttttccggc gtggcggctc gctgcgtatg ttgttggatg 8520 acgatgttac acagccgctt aatactctgt atcgctatgc cacgcagctt atggaggtaa 8580 aagaattcgc cggcgcagcg cgacttttc aattgctgac gatatatgat gcctggtcat 8640 ttgactactg gtttcggtta ggggaatgct gccaggctca aaaacattgg ggggaagcga 8700 tatacgctta tggacgcgcg gcacaaatta agattgatgc gccgcaggcg ccatgggccg 8760 cagcggaatg ctatctcgcg tgtgataacg tctgttatgc aatcaaagcg ttaaaggccg 8820 tggtgcgtat ttgcggcgag gtcagtgaac atcaaattct ccgacagcgt gcagaaaaga 8880 tgttacagca actttctgac aggagctaaa aatgaatcga attcacagta atagcgacag 8940 cgccgcagga gtaaccgcct taacacatca tcacttaagc aatgtcagtt gcgtttcctc 9000 gggttcgctg ggaaagcgcc agcatcgtgt gaattctact tttggcgatg gcaacgccgc 9060 gtgtctgcta tccgggaaaa ttagtcttca ggaggcaagc aatgcgttga agcaactgct 9120 tgatgccgta cccggaaatc ataagcgtcc atcattgcct gactttttgc agaccaatcc 9180 cgcggtttta tcaatgatga tgacgtcatt aatactcaac gtctttggta ataacgctca 9240 atcgttatgc caacagcttg agcgggcaac tgaggtgcaa aatgcattac gtaataagca 9300 ggtaaaggag tatcaggagc agatccagaa agcgatagag caggaggata aagcgcgtaa 9360 agcgggtatt tttggcgcta ttttgactg gattaccggc atattgaaa ccgtgattgg 9420 cgccttaaaa gttgtggaag gttttctgtc cggaaatccc gcagaaatgg ctagcggcgt 9480 agcttatatg gccgcaggtt gtgcaggaat ggttaaagcc ggagccgaaa cggcaatgat 9540 gtgcggtgct gaccacgata cctgtcaggc aattattgac gtgacaagta agattcaatt 9600 tggttgtgaa gccgtcgcgc tggcactgga tgttttccag attggccgtg cttttatggc 9660 gacgagaggt ttatctggcg cagctgcaaa agtgcttgac tccggttttg gcgaggaagt 9720 ggttgagcgt atggtaggtg caggggaagc agaaatagag gagttggctg aaaagttttgg 9780 cgaagaagtg agcgaaagtt tttccaaaca atttgagccg cttgaacgtg aaatggctat 9840 ggcgaatgag atggcagagg aggctgccga gttttctcgt aacgtagaaa ataatatgac 9900 gcgaagcgcg ggaaaaagct ttacgaaaga gggggtgaaa gccatggcaa aagaagcggc 9960 aaaagaagcc ctggaaaat gtgtgcaaga agtggaag ttcctgttaa aaaaattccg 10020 tataaagtt ctcttcaata tgttcaaaaa atcctgtat gccttactga gggattgttc 10080 atttaaaggc ttacaggcta tcagatgtgc aaccgaggc gccagtcaga tgaatactgg 10140 catggttaac acagaaaag cgaagatcga aaaaaata gaggaattaa taactcagca 10200 acggtttctg gatttcataa tgcaaac agaaaaccag aaaaagatag aaaaaaacg 10260 cttagaggag ctttattagg ggagcggtgc cgcgcttaga gatgtattag attackgattga 10320 tcactatagt agcgttcagg cgagaatagc tggctatcgc gcttaatctg aggataaaaa 10380 tatggaagcg agtaacgtag cactgtatt accagcgcct tccttgttaa caccttctc 10440 cactccatct cccctccgggg agggaatggg tactgaatca atgctctctt tattgatga 10500 tatctggatg aagctaatgg agcttgccaa aaagctgcgc gatatcatgc gcagctataa 10560 cgtagaaaaa caacggctgg cctgggaact gcaagtcaat gttttacaga cgcaatgaa 10620 aacaatgat gaagcgttta gagcatcaat gattactgcg ggtggcgcaa tgttgtcggg 10680 tgtactgacg ataggattag gggccgtagg cggggaaacc ggtcttatag cgggtcaagc 10740 cgtaggccac acagctgggg gcgtcatggg cctgggggct ggtgtagcgc aacgtcaaag 10800 tgatcaagat aaagcgattg ccgacctgca acaaaatggg gcccaatctt ataataaatc 10860 cctgacggaa attatggaga aagcaactga aattatgcag caaatcatcg gcgtggggtc 10920 gtcactggtc acggttcttg ctgaaatact ccgggcatta acgaggtaaa catggtgcaa 10980 gaatagagc aatggttacg tcggcatcag gtgtttactg agcctgcata tttaggggag 11040 accgccatat tacttgggca gcagttata ttatcgcctt acctggtgat ctatcgtatt 11100 gaggcaaaag aaatgattat ttgtgagttc aggcgcctga cgcccgggca acctcgacca 11160 cagcaattgt ttcacttact gggactttta cgcgggatat ttgtgcatca cccgcagtta 11220 acatgtttaa agatgttgat aatcaccgac gttctggatg aaaaaaagc catgctacgc 11280 aggaaattat tgcgcatcct gacagtaatg ggagcgacct ttacacagct tgatggcgat 11340 aactggacag ttttaccgc cgagcatctt atccagcgac gttttaat gaccttcctg 11400 acgtaaatca ttcacgtg aaaataaca tcaatagta tgatgatgaa agagatcag 11460 aaaaaayaa tacccgaaga cattctgaaa cagctatt ccgttgatcc ggaaccgtt 11520 tatgccagtg gttacgccctc atggcaggag ggggattatt cgcgcgccgt atcgatttt 11580 agttggctgg tgatggccca gccatggagt tggcgtgccc atattgcatt ggctggcacc 11640 tggatgatgc ttaaagaata cacgacggcc attaatttct atggacatgc cttgatgctg 11700 gatgccagcc atccagacc gttttacca acggggcgtct gtctcaaat gatgggggaa 11760 cccggggttgg cgagagaggc tttcaacc gcaatcaaga tgagttatgc ggatgcctca 11820 tggagtgaga ttcgccagaa tgcgcaata atggttgata ctcttattgc ttaaataaca 11880 gaacgaaata tgaaaatttca tattccgtca gcggcaagta atatagtcga tggtaatagt 11940 cctccttccg atacaagc gaaggaggta tcgttccctc cccctgaat tccagcgcct 12000 ggcaccccg cagcccctgt gctgcttacg cctgaacaaa taggcagca gaggattat 12060 gcgatacatt ttatgcaata cactattcgt gcgctgggtg cgacagtcgt gtttgggtta 12120 tcggttgctg cagcggtaat ttctggcggg gcaggattac ccattgctat tcttgcgggg 12180 gcggcgctcg tgattgctat tggggatgct tgctgtgcgt atcataatta tcaatcgata 12240 tgtcagcaaa aggagccatt acaaaccgcc agtgatagcg ttgctcttgt ggtcagtgcg 12300 ctggccttaa aatgtggggc aagtcttaac tgcgctaaca cccttgctaa ttgtctttct 12360 ttattaatac gttcaggaat cgctatttct atgttggttt tacccctaca gtttccactg 12420 cccgcggctg aaaatattgc ggcctctttg gacatgggga gtgtaattac ctccgttagc 12480 ctgacggcga taggtgcggt actggattat tgccttgccc gcccctctgg cgacgatcag 12540 gaaaattctg ttgatgaact tcatgccgat cccagtgtgt tattggcgga acaaatggca 12600 gcgctctgtc aatctgctac tacacctgca ttaatggaca gttctgatca tacatctcgg 12660 ggagaaccat gaaacctgtt agcccaaatg ctcaggtagg agggcaacgt cctgttaacg 12720 cgcctgagga atcacctcca tgtccttcat tgccacatcc ggaaaccaat atggagagtg 12780 gtagaatagg acctcaacaa ggaaaagagc gggtattggc cggacttgcg aaacgagtga 12840 tagagtgttt tccaaaagaa atttttagtt ggcaaacggt tattttgggc ggacagattt 12900 tatgctgttc cgctggaata gcattaacag tgctaagtgg tggaggcgcg ccgctcgtag 12960 ccctggcagg gattggcctt gctattgcca tcgcggatgt cgcctgtctt atctaccatc 13020 ataaacatca tttgcctatg gctcacgaca gtataggcaa tgccgttttt tatattgcta 13080 attgtttcgc caatcaacgc aaaagtatgg cgattgctaa agccgtctc ctgggcggta 13140 gattagcctt aaccgcgacg gtaatgactc attcatactg gagtggtagt ttgggactac 13200 agcctcattt attagagcgt cttaatgata ttacctatgg actaatgagt tttactcgct 13260 tcggtatgga tgggatggca atgaccggta tgcaggtcag cagcccatta tatcgtttgc 13320 tggctcaggt aacgccagaa caacgtgcgc cggagtaatc gttttcaggt atataccgga 13380 tgttcattgc tttctaaatt ttgctatgtt gccagtatcc ttacgatgta tttattttaa 13440 ggaaaagcat tatggatatt gcacaattag tggatatgct ctcccacatg gcgcaccagg 13500 caggccaggc cattaatgac aaaatgaatg gtaatgattt gctcaaccca gaatcgatga 13560 ttaaagcgca atttgcctta cagcagtatt ctacatttat taattacgaa agttcactga 13620 tcaaaatgat caaggatatg cttagtggaa tcattgctaa aatctgaagt tattagcgac 13680 gatgttcgac ggttgctgct ggaaatcatg tttgcgggcg ttaaccatag cctgatttcc 13740 caggtacatg cgatgttacc agcgctaacg gttattgttc cggataaaaa attacagttg 13800 gtatgtctgg cattattgtt ggcgggttta aatgagccgc taaaagccgc gaaaatttta 13860 tcggatatag atttgccaga ggctatggcg ctgcgtctgt tatttcctgc accaaatgag 13920 gggtttgaaa attgaatatt tctgatatga gcgtagtgcc tgtaagcact caatcttatg 13980 taaagtcctc tgcagaaccg agccaggagc aaattaattt tttgaacaa ttgctgaaag 14040 atgaagcatc caccagtaac gccagtgctt tattaccgca ggttatgttg accagacaaa 14100 tggattatat gcagttaacg gtaggcgtcg attatcttgc cagaatatca ggcgcagcat 14160 cgcaagcgct taataagctg gataacatgg catgaaggtt catcgtatag tatttcttac 14220 tgtccttacg ttctttctta cggcatgtga tgtggatctt tatcgctcat tgccagaaga 14280 tgaagcgaat caaatgctgg cattacttat gcagcatcat attgatgcgg aaaaaaaaca 14340 ggaagaggat ggtgtaacct tacgtgtcga gcagtcgcag tttattaatg cggttgagct 14400 acttagactt aacggttatc cgcataggca gtttacaacg gcggataaga tgtttccggc 14460 taatcagtta gtggtatcac cccaggaaga acagcagaag attaatttt taaaagaaca 14520 aagaattgaa ggaatgctga gtcagatgga gggcgtgatt aatgcaaaag tgaccattgc 14580 gctaccgact tatgatgagg gaagtaacgc ttctccgagc tcagttgccg tatttataaa 14640 atattcacct caggtcaata tggaggcctt tcgggtaaaa attaaagatt tatagagat 14700 gtcaatccct gggttgcaat acagtaagat tagtatcttg atgcagcctg ctgaattcag 14760 aatggtagct gacgtacccg cgagacaaac attctggatt atggacgtta tcaacgccaa 14820 taaagggaag gtggtgaagt ggttgatgaa atacccttat ccgttgatgt tatcgttgac 14880 aggactgtta ttaggagtgg gcatcctgat cggctatttt tgcctgagac gccgtttttg 14940 agccgacctg atcccgaggt gttgcaactt tatcgttatt tctggcaacc tgctcgttac 15000 gctgtaccgg aatggctgga taagctgggc tttcatcttt caaactgctg gcgttatggc 15060 gatcggcccg agttggatcg tcttcttgac agagcgttaa atagactaag aggaagctct 15120 gttattccag cctgtttaaa tgacaggcaa aaacggcagg ttcgtcttgc gccgcgtata 15180 tcggcatttg cctttgggct gggattattc aaactcaggt gtagtgacta ttttatgcta 15240 ccagagtatc ggcaattgct tctacagtgg tttagcgagg atgagatctg gcagctatat 15300 ggttggttgg ggcaaagaga tggcaaatta cttcctccgc aagtgatgca acaaactgca 15360 ttgcagatcg gtaccgccat tcttaatcgg gaagcgcatg acgatgcggt tttacatgcg 15420 ctattagtat tattaccccc tccgcagcgt atactttggc cgaagacttc tcttaccgag 15480 attatcttca tggagttt gctatgagtt ttacttcact tcctctgacg gaaatttacc 15540 ataagctacc cgctcgaat attattgagt cacagtggat aacattaca tattactttat 15600 ttgcgcaaga gcaacaagct agagagttt cacatgctat tgtgagctcc gcttaccgta 15660 aggctgaaaa atcatccga gacgcctatc gttatcagcg tgaacagaaa gttgagcagc 15720 aacaagaact agcgtgcttg cgtaaaaata cgctggaaa atggaagtg gatggctgg 15780 aacagcatgt aaaacattta caacacgatg aaatcatt tcgttcattg gtcgatcacg 15840 cagcgcatca tattaaaaat agtatagaac aggttctgtt ggcctggttc gaccacagt 15900 cggtagacag tgttatgtgc catcgtctgg cacgccaggc cacggctatg gcggaagagg 15960 gagcgcttta tttgcgtatt catcctgaaa aagaggcatt gatgcgagaa acttttggca 16020 agcggtttac gttgattatc gagcctggtt tctctcccga tcaggctgaa ctttcctca 16080 cacgatatgc cgttgaattt tcactctc gtcattca cgcgttactg aaatggttac 16140 gtaatggtga agataaaga ggtagcgatg atattaaaa ttaatgagat aaaatgacg 16200 ccccctacag catttacccc tggccaggtt atagaggaac aagaggttat ttcgccttca 16260 atgttagctc tccaggagtt acaggaaacg acgggggcag cgctctatga gacgatggaa 16320 gaaataggaa tggcgctgag tggtaaactg cgcgaaaatt ataaattcac tgatgctgag 16380 aaactggagc gcagacagca ggctttgctg cgtttgataa aaaaataca ggaggataat 16440 ggggcaacgt tgcgtccgct taccgaagag aatagtgatc ctgatttaca gaatgcgtat 16500 caaattatcg ctcttgcaat ggcgcttact gccggcgggt tgtcaaaaaa gaaaaaacgc 16560 gatttgcaat cgcaactgga tacgcttaca gcggaggagg gatgggaact tgccgttttt 16620 agtttactgg aacttggcga agtggatacc gctacgctgt cctctctgaa gcgttttatg 16680 caacaggcga tagacaacga tgaaatgccc ttatcgcagt ggttcagacg cgtggcagac 16740 tggccggatc gctgtgaacg ggtccgtatt ttgctaagag cagtagcctt tgaacttagc 16800 atatgcatcg aaccctcgga gcaaagtcgt ttggccgcag cattagtacg tttgcgtcgt 16860 ttgctgttat tccttggcct tgaaaaagag tgccagcgtg aggagtggat ttgccagttg 16920 ccgcctaata cattactgcc gctactactc gatattattt gtgagcgctg gcttttcagt 16980 gattggttgc ttgatagact taccgctata gtttcttcat cgaagatgtt caatcggtta 17040 ctccaacaac ttgatgcgca gtttatgctg atacccgata actgttttaa cgacgaagat 17100 caacgtgaac aaattctcga aacgcttcgt gaagtaaaga taaatcaggt tttattctga 17160 tacctggctt tcaatattta ggtaaattgg ctttctggct catcatgagg cgtcaggatg 17220 gattgggatc tcattactga acgtaatatt cagcttttta ttcaattagc aggattagct 17280 gaacggcctt tagcaaccaa tatgttctgg cggcaaggac aatatgaaac ctatctaaac 17340 tatcataacg gtcgtattca cttatgtcag atactcaagc aaaccttctt agacgaagaa 17400 ctgcttttta aagcgttggc taactggaaa cccgcagcgt tccagggtat tcctcaacga 17460 ttatttttgt tgcgcgatgg gcttgcaatg agttgttctc cacctctttc cagctccgcc 17520 gagctctggt tacgattaca tcatcgacaa ataaaatttc tggagtcgca atgcgttcat 17580 ggttaggtga gggagtcagg gcgcaacagt ggctcagtgt atgcgcgggt cggcaggata 17640 tggttctggc gacggtgtta ttaatcgcta ttgtgatgat gctgttacct ttgccgacct 17700 ggatggttga tatcctgatt actatcaacc ttatgttttc agtgatcctg ctcttaattg 17760 ctatttatct tagtgaccct ctcgatttat cggtatttcc gtctttatta cttattacta 17820 cattatatcg tttgtcactc acaatcagca catcacggct ggtactgtta caacataatg 17880 ccggtaatat tgtggatgct ttcggtaagt ttgtcgtagg aggaaatctc accgttgggt 17940 tggtcgtatt taccatcatt actatcgtgc aatttattgt cattacaaaa ggtatcgaga 18000 gggtggcgga agttagcgca cgtttctcgc ttgatgggat gccaggcaaa caaatgagta 18060 tcgatggcga tttgcgtgcc ggagttatcg atgcagacca tgcccgtaca ttaagacagc 18120 atgtccagca ggaaagccgc tttctcggtg cgatggacgg tgcgatgaaa tttgttaaag 18180 gcgatacgat tgccggtatt attgttgttc tggtgaacat tatcggcggt atcattatcg 18240 ctatcgtaca atatgatatg tcgatgagtg aggctgttca cacttatagc gtactgtcaa 18300 tcggagatgg tttatgtggg caaattccat cgctgctgat ttcccttagc gcgggaatta 18360 ttgtcacccg tgtcccgggt gagaaacgcc agaacctggc gacagagttg agttctcaaa 18420 ttgccagaca acctcagtcg ctcatattaa ccgctgtggt tttaatgctc ctcgctttaa 18480 ttcctggctt tccttttatc actctcgctt tcttttcagc gttgttagca ttgccaatta 18540 tcctcattcg ccgcaaaaag tctgtggttt ccgcaaatgg cgtcgaagca ccggaaaaag 18600 atagtatggt tcccggcgca tgtcctctaa tcttacgtct tagcccgacg ttacattctg 18660 ccgacctgat tcgtgatatt gacgccatga gatggttttt atttgaggat accggcgtcc 18720 ctctccctga ggtgaatatt gaggttttgc ctgaacccac cgaaaaattg acggtactgc 18780 tatatcagga acccgtattt agtttatcta ttcccgctca ggcggattat ttattgatag 18840 gcgcggacgc tagtgtggtg ggtgacagcc agacgttacc gaacgggatg gggcagatct 18900 gttggcttac aaaagacatg gcccataagg cgcaaggttt tggactggac gttttcgcgg 18960 gcagccaacg tatctctgcc ttattaaaat gtgtcctgct tcggcatatg ggagagttta ttggtgttca ggaaacgcgt tatctaatga atgcgatgga aaaaaactac tctgagctgg tgaaagagct tcagcgccag ttacccatta ataaaatcgc tgaaactttg caacggcttg tatcagagcg ggtttctatt agagatttac gtcttatttt cggcacctta attgactggg 19200 cgccacgtga aaaagatgtc ctgatgttga cagaatgt ccgtatcgcg cttcgtcgtc atattctgcg tcgtcttaat ccggaagga aaccgctgcc gattttgcgg atcggcgaag gtattgaaaa cctcgtgcgt gaatccattc gccagacggc aatggggacc tatactgcgc 19380. tgtcgtctcg tcataagacg cagatcctgc aacttatcga gcaggcgctg aagcagtcag 19440 ccaattatt cattgtcact tctgtcgaca cccgacgttt cttgcgaaaa attack ccaccttgtt cgacgtaccg attttgtcat ggcaggaatt aggagaggag agccttatac 19560 aagtggtaga aagtattgac cttagcgaag aggagttggc ggacaatgaa gaatgaattg atgcaacgtc tgaggctgaa atatccgccc cccgatggtt attgtcgatg gggccgaatt 19680 caggatgtca gcgcaacgtt gttaaatgcg tggttgcctg gggtatttat gggcgagttg 19740 tgctgtataa agcctggaga agaacttgct gaagtcgtgg ggattaatgg cagcaaagct 19800 ttgctatctc cttttacgag tacaatcggg cttcactgcg ggcagcaagt gatggcctta 19860 aggcgacgcc atcaggttcc cgtgggcgaa gcgttattag ggcgagttat tgatggcttt 19920 ggtcgtcccc ttgatggccg cgaactgccc gacgtctgct ggaaagacta tgatgcaatg 19980 cctcctcccg caatggttcg acagcctatc actcaaccat taatgacggg gattcgcgct 20040 attgatagcg ttgcgacctg tggcgaaggg caacgagtgg gtattttttc tgctcctggc 20100 gtggggaaaa gcacgcttct ggcgatgctg tgtaatgcgc cagacgcaga cagcaatgtt 20160 ctggtgttaa ttggtgaacg tggacgagaa gtccgcgaat tcatcgattt tacactgtct 20220 gaagagaccc gaaaacgttg tgtcattgtt gtcgcaacct ctgacagacc cgccttagag 20280 cgcgtgaggg cgctgtttgt ggccaccacg atagcagaat tttttcgcga taatggaaag 20340 cgagtcgtct tgcttgccga ctcactgacg cgttatgcca gggccgcacg ggaaatcgct 20400 ctggccgccg gagagaccgc ggtttctgga gaatatccgc caggcgtatt tagtgcattg 20460 ccacgacttt tagaacgtac gggaatggga gaaaaaggca gtattaccgc attttatacg 20520 gtactggtgg aaggcgatga tatgaatgag ccgttggcgg atgaagtccg ttcactgctt 20580 gatggacata ttgtactatc ccgacggctt gcagagagg ggcattatcc tgccattgac 20640 gtgttggcaa cgctcagccg cgtttttcca gtcgttacca gccatgagca tcgtcaactg 20700 gcggcgatat tgcgacggtg cctggcgctt taccaggagg ttgaactgtt aatacgcatt 20760 ggggaatacc agcgaggagt tgatacagat actgacaaag ccattgatac ctatccggat 20820 atttgcacat ttttgcgaca aagtaaggat gaagtatgcg gacccgagct acttatagaa 20880 aaattacacc aaatactcac cgagtgatca tggaaacttt gctgggagata atcgcgcggc 20940 gtgaaaagca attacgcggc aagcttaccg tacttgatca gcagcaacag gcgattatta 21000 cggaacagca gatttgccag acgcgcgctt tagcagtgtc taccagactg aaagaattaa 21060 tgggctggca aggtacgtta tcttgtcatt tattgttgga taagaaacaa caaatggccg 21120 ggttattcac tcaggcgcag agctttttga cgcaacggca gcagttagag aatcagtatc 21180 agcagcttgt ctcccggcga agcgaattac agaagaattt taatgcgctt atgaaaaaga 21240 aagaaaaaat tactatggta ttaagcgatg cgtattacca aagttgaggg aagtcttggg 21300 ttgccatgcc agtcttatca ggatgataac gaggcggagg cggaacgtat ggactttgaa 21360 caactcatgc accaggcatt acccattggt gagaataatc ctcctgcagc attgaataag 21420 aacgtggtt tcacgcaacg ttatcgtgtt agtggcggtt atcttgacgg tgtagagtgt 21480 gaagtatgtg aatcaggggg gctaatccag ttaagaatca atgtccctca tcatgaaatt 21540 taccgttcga tgaaagcgct aaagcagtgg ctggagtctc agttgctgca tatggggtat 21600 ataatttccc tggagatatt ctatgttaag aatagcgaat gaagagcgtc cgtgggtgga 21660 gatacttcca acgcaaggcg ctaccattgg tgagctgaca ttgagtatgc aacaatatcc 21720 agtacagcaa gggacattat ttaccataaa ttatcataat gagctgggta gggtgtggat 21780 tgcagaacaa tgctggcagc gctggtgtga agggctaatt ggcaccgcta atcgatcggc 21840 tatcgatcct gaattgctat atggaatagc tgaatggggg ctggcgccgt tattgcaagc 21900 cagtgatgca accctctgtc agaacgagcc gccaacatcc tgcagtaatc taccacatca 21960 gctagcgttg catattaaat ggacagttga agagcatgag ttccatagca ttatttttac 22020 atggccaacg ggttttttgc gcaatatagt cggagagctt tctgctgagc gacaacagat 22080 ttatcctgcc cctcctgtgg tagtccctgt atattcaggc tggtgccagc ttacattaat 22140 cgaacttgag tctatcgaaa tcggcatggg cgttcggatt cattgcttcg gcgacatcag 22200 actcggtttt tttgctattc aactacctgg gggaatctac gcaagggtgt tgctgacaga 22260 ggataacacg atgaaatttg acgaattagt ccaggatatc gaaacgctac ttgcgtcagg 22320 gagcccaatg tcaaagagtg acggaacgtc ttcagtcgaa cttgagcaga taccacaaca 22380 ggtgctcttt gaggtcggac gtgcgagtct ggaattgga caattacgac aacttaaaac gggggacgtt ttgcctgtag gtggatgttt tgcgccagag gtgacgataa gagtaaatga 22500. ccgtattatt gggcaaggtg agttgattgc ctgtggcaat gaatttatgg tgcgtattac acgttggtat ctttgcaaaa atacagcgta aacctgata gaaaaata atgcgaacaa fathercg ttccaggtcg tgtcatgaga gatacagtat gtctttaccc gattcgcctt tgcaactgat tggtatattg tttctgcttt caatactgcc tctcattatc gtcatgggaa cttctttcct taaactggcg gtggtatttt cgattttacg aaatgctctg ggtattcaac aagtcccccc aaatatcgca ctgtatggcc ttgcgcttgt actttcctta ttcattatgg 22860 ggccgacgct attagctgta aaagagcgct ggcatccggt tcaggtcgct ggcgctcctt 22920 tctggacgtc tgagtgggac agtaaagcat tagcgcctta tcgacagttt ttgcaaaaaa actctgaga gaggagcc aattattttc ggaatttgat aaaacgaacc tggcctgag acataaaaag aaagataaaa cctgattctt tgctcatatt aattccggca tttacggtga gtcagttaac gcaggcattt cggattggat tacttattta tcttcccttt ctggctattg 23160 acctgcttat ttcaaatata ctgctggcta tggggatgat gatggtgtcg ccgatgacca 23220 tttcattacc gtttaagctg ctaatatttt tactggcagg cggttgggat ctgacactgg 23280 cgcaattggt acagagcttt tcatgaatga ttctgaattg acgcaatttg taacgcaact 23340 tttatggatc gtccttttta cgtctatgcc ggtagtgttg gtggcatcgg tagttggtgt 23400 catcgtaagc cttgttcagg ccttgactca aatacaggac caaacgctac agttcatgat 23460 taaattattg gcaattgcaa taaccttaat ggtcagctac ccatggctta gcggtatcct 23520 gttgaattat acccggcaga taatgttacg aattggagag catggttgaa tggcacaaca 23580 ggtaaatgag tggcttattg cattggctgt ggcttttatt cgaccattga gcctttcttt 23640 attacttccc ttattaaaaa gtggcagttt aggggccgca cttttacgta atggcgtgct 23700 tatgtcactt acctttccga tattaccaat catttaccag cagaagatta tgatgcatat 23760 tggtaaagat tacagttggt tagggttagt cactggagag gtgattattg gtttttcaat 23820 tgggttttgt gcggcggttc ccttttgggc cgttgatatg gcggggtttc tgcttgatac 23880 tttacgtggc gcgacaatgg gtacgatatt caattctaca atagaagctg aaacctcact 23940 ttttggcttg cttttcagcc agttcttgtg tgttattttc tttataagcg gcggcatgga 24000 gtttatatta aacattctgt atgagtcata tcaatattta ccaccagggc gtactttatt 24060 atttgaccag caatttttaa aatatatcca ggcagagtgg agaacgcttt atcaattatg 24120 tatcagcttc tctcttcctg ccataatatg tatggtatta gccgatctgg ctttaggtct 24180 tttaaatcgg tcggcacaac aattgaatgt gtttttcttc tcaatgccgc tcaaaagtat 24240 attggttcta ctgacgctcc tgatctcatt cccttatgct cttcatcact atttggttga 24300 aagcgataaa ttttatattt atctaaaaga ctggtttcca tctgtatgag cgagaaaaca 24360 gaacagccta cagaaaagaa attacgtgat ggccgtaagg aagggcaggt tgtcaaaagt 24420 attgaaataa catcattatt tcagctgatt gcgctttatt tgtattttca tttctttact 24480 gaaaagatga ttttgatact gattgagtca ataactttca cattacaatt agtaaataaa 24540 ccatttctt atgcattaac gcaattgagt catgctttaa tagagtcact gacttctgca 24600 ctgctgtttc tgggcgctgg ggtaatagtt gctactgtgg gtagcgtgtt tcttcaggtg 24660 ggggtggtta ttgccagcaa ggccattggt tttaaaagcg agcatataaa tccggtaagt 24720 aattttaagc agatattctc tttacatagc gtagtagaat tatgtaaatc cagcctaaaa 24780 gttatcatgc tatctcttat ctttgccttt ttcttttatt attatgccag tacttttcgg 24840 gcgctaccgt actgtgggtt agcctgtggc gtgcttgtgg tttcttcttt aataaaatgg 24900 tttgggtag gggtgatggt ttttatatc gtcgttggca tactggacta ttcttttcaa 24960 tattataaga ttagaaaaga tctaaaaatg agtaaagatg acgtaaaaca ggagcataaa 25020 gatctggagg gcgaccctca aatgaagacg cggcgtcggg aaatgcagag tgaaatacaa 25080 agtgggagtt tagctcaatc tgttaaacaa tctgttgcgg tagtgcgtaa tccaacgcat 25140 attgcggttt gtcttggcta tcatcccacc gatatgccaa taccacgcgt cctggaaaaa 25200 ggcagtgatg ctcaagctaa ctatattgtt aacatcgctg aacgcaactg catccccgtt 25260 gttgaaaatg ttgagctggc ccgctcatta ttttttgaag tggaacgcgg agataaaatt 25320 cctgaaacgt tatttgaacc cgttgcagcc ttgttacgta tggtgatgaa gatagattat 25380 gcgcattcta ccgaaacacc ataaatgctt ttggtatgct tcttcaggcc actgcgaagg 25440 25440 <210> 3 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> hilD forward primer <400> 3 tttgtaagta atagtcatca gcgtcctgcc gcagataact tgtaggctgg agctgcttc 59 <210> 4 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> hilD reverse primer <400> 4 agcttccact gtctactggg tgacgaagat ataatgttgt catatgaata tcctcctta 59 <210> 5 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> SPI-1 forward primer <400> 5 taattatatc atgatgagtt cagccaacgg tgatatggcc tgtaggctgg agctgcttc 59 <210> 6 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> SPI-1 reverse primer <400> 6 gctggaagga tttcctctgg caggcaacct tataatttca catatgaata tcctcctta 59 <210> 7 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> SPI-2 forward primer <400> 7 tgcttaatac catcggacgc ccctggttaa tactctatta tgtaggctgg agctgcttc 59 <210> 8 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> SPI-2 reverse primer <400> 8 ctgaagaagc ataccaaaag catttatggt gtttcggtag catatgaata tcctcctta 59 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> EcoRI (pJL39 plasmid) forward primer <400> 9 cggaattcac catgtctaga ttagataaaa gtaaagtgat taacag 46 <210> 10 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> PvuII-XbaI (pJL39 plasmid) reverse primer <400> 10 gctctagaca gctgttaaga cccactttca catttaagtt gtttttct 48 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> pSF-OXB1 forward primer <400> 11 ctactccgtc aagccgtcaa gctgttgtga ccgcttgct 39 <210> 12 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> pSF-OXB1 reverse primer <400> 12 tgaattcctc ctgctagcta gttggtaacg aatcagacgc cgggtaatac cggatag 57 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Cytolysin A forward primer <400> 13 agtccatggt tatgaccgga atatttgc 28 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Cytolysin A reverse primer <400> 14 gatgtttaaa ctcagacgtc aggaacctc 29 <210> 15 <211> 912 <212> DNA <213> unknown <220> <223> Cytolysin A gene <400> 15 atgaccggaa tatttgcaga acaaactgta gaggtagtta aaagcgcgat cgaaaccgca 60 gacggggcat tagatcttta taacaaatac ctcgaccagg tcatcccctg gaagaccttt 120 gatgaaacca taaaagagtt aagccgtttt aaacaggagt actcgcagga agcttctgtt 180 ttagttggtg atattaaagt tttgcttatg gacagccagg acaagtattt tgaagcgaca 240 caaactgttt atgaatggtg tggtgtcgtg acgcaattac tctcagcgta tattttacta 300 tttgatgaat ataatgagaa aaaagcatca gcccagaaag acattctcat taggatatta 360 gatgatggtg tcaagaaact gaatgaagcg caaaaatctc tcctgacaag ttcacaaagt 420 ttcaacaacg cttccggaaa actgctggca ttagatagcc agttaactaa tgatttttcg 480 gaaaaaagta gttatttcca gtcacaggtg gatagaattc gtaaggaagc ttatgccggt 540 gctgcagccg gcatagtcgc cggtccgttt ggattaatta tttcctattc tattgctgcg 600 ggcgtgattg aagggaaatt gattccagaa ctgaataaca ggctaaaaac agtgcaaaat 660 ttctttacta gcttatcagc tacagtgaaa caagcgaata aagatatcga tgcggcaaaa 720 ttgaaattag ccactgaaat agcagcaatt ggggagataa aaacggaaac cgaaacaacc 780 agattctacg ttgattatga tgatttaatg ctttctttat taaaaggagc tgcaaagaaa 840 atgattaaca cctgtaatga ataccaacaa agacacggta agaagacgct tttcgaggtt 900 cctgacgtct aa 912

Claims

1. A mutant strain of Salmonella for use against tumors, wherein Salmonella pathogenicity island-1 (SPI-1) and Salmonella pathogenicity island-2 (SPI-2) are deleted; and the mutant strain of Salmonella is a strain in which the Salmonella-relA gene encoding ppGpp synthetase and the Salmonella-spoT gene are inactivated, and wherein the mutant strain of Salmonella has a specific and selective targeting ability against cancer by inhibiting tumor growth and having reduced viability in normal liver; wherein the Salmonella mutant strain is derived from Salmonella typhimurium (ATCC® 19525™) Salmonella typhimurium ).

2. The mutant strain of Salmonella according to claim 1, wherein the Salmonella pathogenicity island-1 (SPI-1) consists of the nucleotide sequence set forth in SEQ ID NO:

1.

3. The mutant strain of Salmonella according to claim 1, wherein Salmonella pathogenicity island-2 (SPI-2) consists of the nucleotide sequence set forth in SEQ ID NO:

2.

4. The mutant strain of Salmonella according to claim 1, wherein the mutant strain of Salmonella is a strain into which a gene encoding an anticancer protein is additionally introduced.

5. The mutant strain of Salmonella according to claim 4, wherein the anticancer protein is at least one selected from the group consisting of a toxin protein, a cancer antigen-specific antibody or antibody fragment, a tumor suppressor protein, an angiogenesis inhibitor, a cancer antigen, a prodrug-converting enzyme, and a pro-apoptotic protein.

6. The mutant strain of Salmonella according to claim 5, wherein the toxin protein is at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA).

7. The mutant strain of Salmonella according to claim 6, wherein cytolysin A consists of the nucleotide sequence set forth in SEQ ID NO:

15.

8. The mutant strain of Salmonella according to claim 5, wherein the tumor suppressor protein is at least one selected from the group consisting of retinoblastoma (RB) protein, p53 protein, adenomatous polyposis coli (APC) protein, phosphatase and tensin homolog (PTEN), and cyclin-dependent kinase inhibitor 2A (CDKN2A) protein.

9. The mutant strain of Salmonella according to claim 5, wherein the angiogenesis inhibitor is at least one selected from the group consisting of angiostatin, endostatin, thrombospondin, and proteinase inhibitor protein.

10. The mutant strain of Salmonella according to claim 5, wherein the cancer antigen is at least one selected from the group consisting of alpha-fetoprotein (AFP), vascular endothelial growth factor receptor 2 (VEGFR2), Survivin, Legumain, and prostate cancer specific antigen (PCSA).

11. The Salmonella mutant strain of claim 5, wherein the prodrug converting enzyme is at least one selected from the group consisting of thymidine kinase, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, carboxypeptidase G2, chromate reductase YieF, herpes simplex virus type 1 thymidine kinase / ganciclovir (HSV1-TK / GCV), and beta-glucuronidase.

12. The Salmonella mutant strain of claim 5, wherein the pro-apoptotic protein is L-ASNase or RNA-binding motif protein 5 (RBM5).

13. The Salmonella mutant strain of claim 1, wherein the Salmonella mutant strain is a mutant strain lacking the ability to synthesize guanosine polyphosphate.

14. A method for producing a Salmonella mutant strain for use against tumors, the method comprising knocking out the Salmonella pathogenicity island-1 SPI-1 and Salmonella pathogenicity island-2 SPI-2 genes from a Salmonella strain, and further comprising the step of inactivating a ppGpp synthetase-encoding gene to obtain a transformed strain; wherein the ppGpp synthetase-encoding gene is a Salmonella-relA gene and a Salmonella-spoT gene; wherein the Salmonella mutant strain is derived from Salmonella typhimurium (ATCC® 19525™) Salmonella typhimurium ).

15. The method of claim 14, wherein, Knocking out the Salmonella pathogenicity island-1 SPI-1 and Salmonella pathogenicity island-2 SPI-2 genes is performed by a recombination vector.

16. The method of claim 14, wherein the Salmonella pathogenicity island-1 SPI-1 consists of the nucleotide sequence set forth in SEQ ID NO:

1.

17. The method of claim 14, wherein the Salmonella pathogenicity island-2 SPI-2 consists of the nucleotide sequence set forth in SEQ ID NO:

2.

18. The method of claim 14, further comprising the step of introducing a gene encoding an anticancer protein.

19. The method of claim 18, wherein the anticancer protein is at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and cytolysin A (ClyA).

20. The method of claim 19, wherein the gene encoding cytolysin A consists of the nucleotide sequence set forth in SEQ ID NO:

15.

21. The method of claim 14, wherein the Salmonella strain is a mutant strain lacking the ability to synthesize guanosine polyphosphate.

22. The method of claim 14, further comprising the step of culturing the transformed strain.

23. The method of claim 14, further comprising the step of culturing the transformed strain in an antibiotic medium and selecting the mutant strain.

24. A pharmaceutical composition for treating cancer, the pharmaceutical composition containing as an active ingredient the Salmonella mutant strain of claim 1.

25. The pharmaceutical composition of claim 24, wherein the cancer is selected from the group consisting of blood cancer, endocrine gland cancer.

26. The pharmaceutical composition of claim 24, wherein the cancer is selected from the group consisting of small intestine cancer, lymph gland cancer, thyroid cancer, biliary tract cancer, colorectal cancer, brain tumor, uterine cancer, skin cancer, leukemia.

27. The pharmaceutical composition of claim 24, wherein the cancer is selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, esophageal cancer, gallbladder cancer, oral cancer, liver cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, lung cancer, undifferentiated thyroid cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone / soft tissue sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, and solitary myeloma.

Images