A strain of Pseudomonas aeruginosa phage and its application

Abstract

The present invention provides a Pseudomonas aeruginosa phage and its application. The name of the Pseudomonas aeruginosa phage is phage J3, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 16, 2024, with a deposit number of GDMCC No: 64627-B1, and a taxonomic name: Pseudomonas aeruginosa phage. Phage J3 can not only enrich the germplasm resource library of Pseudomonas aeruginosa phage, but also provide an optional basic material for the biological control of Pseudomonas aeruginosa. In a medical environment where drug-resistant bacteria are prevalent and common disinfectants are ineffective, phage J3 has a high safety factor and a wide range of applications in the preparation of phage disinfectants or phage preparations for medical device surfaces, hospital environments and other environments, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a Pseudomonas aeruginosa phage and application thereof. Background Art

[0002] Pseudomonas aeruginosa (PA) is a conditional pathogen that can cause a series of infectious diseases in humans and is also a common pathogen in hospitals. When the host defense system is damaged, resulting in a decrease in the body's immune function, Pseudomonas aeruginosa infection is prone to occur. Since the infection symptoms caused by this pathogen are prone to recurrence and the treatment time is long, it is difficult to completely cure it. Antibiotics are often used clinically to achieve the effect of treatment and prevention, but studies have shown that up to 90% of Pseudomonas aeruginosa were detected to show resistance to a single antibiotic in clinical isolates, and due to the overuse and abuse of antibiotics, bacteria continue to produce new and more complex resistance mechanisms, and the resistance is enhanced, which greatly threatens human public health. At the same time, the speed at which bacteria develop resistance is much faster than the speed of developing new antibiotics, so there is an urgent need to find new therapies that can replace antibiotics to treat Pseudomonas aeruginosa infections.

[0003] Bacteriophage (phage) is a virus that specifically infects bacteria. It can be isolated from natural environments such as sewage and feces. It is the most numerous and widely distributed organism. The infection mechanism of bacteriophage is to bind to the host bacteria through the tail protein and adsorb to the host bacteria to initiate phage infection. It has strict host specificity and a unique mechanism of action. It has no infection effect on humans, animals and plants, and can target and kill pathogenic host bacteria without affecting the normal flora of the body. Because bacteriophages have strict bactericidal specificity and are not easy to develop drug resistance, phage therapy has once again entered people's field of vision and has the potential to be developed into an antibacterial drug for the treatment of drug-resistant bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide a Pseudomonas aeruginosa phage and its application, to isolate and identify the Pseudomonas aeruginosa phage, to study its biological characteristics and genomic informatics, etc., to use it as a potential phage agent, in order to lay a foundation for the practical application of phage, which is of great significance for understanding phage therapy and overcoming the limitations of phage therapy.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a Pseudomonas aeruginosa phage, named phage J3, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 16, 2024, with a deposit number of GDMCC No: 64627-B1, and a taxonomic name: Pseudomonas aeruginosa phage.

[0007] Furthermore, phage J3 belongs to the family of long-tailed phages and has a polyhedral head; the titer number level is ≥10 10 The growth latent period is 60 minutes, the rapid growth period is 60 minutes to 90 minutes, the temperature stability range is -20℃ to 40℃, and the pH stability range is 6 to 9.

[0008] Furthermore, the sequence of the genomic DNA of bacteriophage J3 is shown in SEQ ID NOs: 1-5.

[0009] The present invention also provides a medicine or biological preparation for preventing or treating Pseudomonas aeruginosa infectious diseases, comprising the above-mentioned Pseudomonas aeruginosa phage.

[0010] Furthermore, the drug or biological preparation uses bacteriophage J3 as the only active ingredient.

[0011] Furthermore, the Pseudomonas aeruginosa includes Pseudomonas aeruginosa PAO1.

[0012] The present invention further provides the use of the above-mentioned Pseudomonas aeruginosa phage in the preparation of drugs or biological preparations for preventing or treating Pseudomonas aeruginosa infectious diseases, such as lysate, phage pharmaceutical preparations, compositions, and combinations of phages and antibiotics.

[0013] The present invention also provides the use of Pseudomonas aeruginosa phage in preparing phage disinfectant or inhibitor, which can be used for sterilization and disinfection of medical equipment environment, hospital environment, animal breeding environment and other environments.

[0014] The beneficial effects of the present invention are:

[0015] The present invention uses Pseudomonas aeruginosa PAO1 as a host, samples are taken from untreated sewage in hospitals, and phage J3 is screened out. The phage can efficiently lyse Pseudomonas aeruginosa PAO1, providing a phage source for the hospital to treat Pseudomonas aeruginosa infection. Phage J3 can not only enrich the germplasm resource bank of Pseudomonas aeruginosa phage, but also provide a selectable basic material for biological control of Pseudomonas aeruginosa. Phage J3 has good temperature tolerance and acid-base tolerance, and can maintain a relatively stable high titer level under long-term storage at room temperature. The phage can show obvious lysis effect on PAO1 within 2 hours, showing good therapeutic effect and application prospects. The phage J3 has no virulence gene through whole genome detection, which ensures the safety and reliability of the phage preparation during use. The open reading frame encoding endolysin is found in the protein function annotation results, which ensures the effectiveness of the phage preparation. In medical environments where drug-resistant bacteria are prevalent and common disinfectants are ineffective, bacteriophage J3 has a high safety factor and a wide range of applications in the preparation of phage disinfectants or phage preparations for use on medical device surfaces, hospital environments, and other environments, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Plaque image of Pseudomonas aeruginosa phage J3.

[0017] Figure 2 Genome map of Pseudomonas aeruginosa phage J3.

[0018] Figure 3 Phylogenetic tree analysis of Pseudomonas aeruginosa phage J3.

[0019] Figure 4 Transmission electron microscopy of Pseudomonas aeruginosa phage J3.

[0020] Figure 5 Lytic kinetics of Pseudomonas aeruginosa phage J3.

[0021] Figure 6 Optimal multiplicity of infection of Pseudomonas aeruginosa phage J3.

[0022] Figure 7 One-step growth curve of Pseudomonas aeruginosa phage J3.

[0023] Figure 8 Temperature stability of Pseudomonas aeruginosa phage J3.

[0024] Fig. 9 pH stability of Pseudomonas aeruginosa phage J3. DETAILED DESCRIPTION

[0025] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0026] The Pseudomonas aeruginosa phage isolated by the present invention is named phage J3, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 16, 2024, with a deposit number of GDMCC No: 64627-B1, and a deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, and a taxonomic name: Pseudomonas aeruginosa phage.

[0027] Example

[0028] 1. Preparation of culture medium

[0029] (1) LB solid medium: add 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, and 15 g agar powder to 1 L ultrapure water, adjust the pH to 7.2-7.4, fully dissolve, and sterilize under high temperature and high pressure for later use.

[0030] (2) LB liquid culture medium: add 5 g yeast powder, 10 g tryptone, and 10 g sodium chloride to 1 L ultrapure water, adjust the pH to 7.2-7.4, fully dissolve and sterilize at high temperature and high pressure for later use.

[0031] 2. Isolation and Purification of Pseudomonas aeruginosa Phage

[0032] Pseudomonas aeruginosa PAO1 was used as the host bacteria, and a single colony was inoculated into LB liquid culture medium and cultured at 37°C until the logarithmic phase. Hospital sewage was centrifuged at 4000rpm for 5 minutes, and the supernatant was filtered with a 0.22μm sterile filter. The treated sewage filtrate was mixed with the logarithmic phase host bacteria and cultured at 37°C overnight. After the culture was completed, the mixed solution was centrifuged at 4000rpm for 5 minutes, and the supernatant was filtered with a 0.22μm sterile filter. The filtrate was used as the phage stock solution. The logarithmic phase bacterial solution was mixed with the phage stock solution, and then the mixed solution was quickly mixed with the LB solid culture medium that was cooled to an appropriate temperature, and the LB solid double-layer plate was poured and cultured at 37°C overnight. If a clear and bright plaque is formed, it proves that the phage has been successfully isolated; otherwise, there is no plaque.

[0033] If plaques appear on the plate, pick a single large and transparent plaque and inoculate it in the logarithmic phase host bacterial solution. After culture, dilute it and purify it again using the double-layer plate method for 3-4 times until plaques of uniform size and shape are formed. This is the purified phage. Pick a single plaque and inoculate it in the logarithmic growth phase host bacterial solution, expand the culture, mix the phage bacterial solution with 60% glycerol in equal amounts to obtain the phage preservation solution (30% glycerol), and store it at -80℃ for later use. The results are as follows Figure 1 The phage can form clear plaques on the double-layer plate with no halo around and clear and regular edges. It is a Pseudomonas aeruginosa phage.

[0034] 3. Extraction and sequencing of bacteriophage J3 genome

[0035] Transfer the phage culture solution treated with 0.5% chloroform to a centrifuge tube, centrifuge at 8000×g for 10min, and remove the precipitated bacterial debris. Add 5μL RNase A and 10μL DNase I to 10mL supernatant, mix thoroughly, and incubate at 37℃ for 30min. Add 4mL phage precipitation solution to the supernatant, shake until dissolved, and incubate at ice bath for 1h or 4℃ overnight. Centrifuge at 10000×g for 20min at 4℃, and discard the supernatant. Add 1mL SM buffer to thoroughly wash the tube wall and precipitate, transfer to a new centrifuge tube, add 40μL phage lysis solution, and incubate at 68℃ for 15min. Add an equal volume of protein removal solution, mix gently, centrifuge at 12000×g for 5min, take the supernatant to a new centrifuge tube, add an equal volume of pre-cooled phage rinse solution, mix gently, incubate at -20℃ for 1h, centrifuge at 12000×g for 10min at 4℃, and discard the supernatant. Add an appropriate amount of 70% ethanol solution and mix well. Centrifuge at 8000×g for 8 min at 4°C, discard the supernatant, and repeat the washing step once. Dry the DNA naturally at room temperature, add an appropriate amount of TE buffer, and store at -20°C.

[0036] The extracted whole genome DNA samples of phage J3 were sent to Shanghai Lingen Biotech Co., Ltd. for sequencing. The sequencing method was based on the second-generation sequencing technology of Illumina Miseq high-throughput sequencing platform. The specific steps were: using the PhageDNAIsolation Kit to extract the total phage DNA, using Illumina sequencing technology to complete the phage genome amplification and deep sequencing, constructing the Illumina PE library, assembling the obtained reads using ABySS, splicing the genome using SPAdes, and annotating the gene function. The results are as follows: Figure 2 , the full genome length of phage J3 is 38008 bp (SEQ ID NO: 1-5):

[0037] GCTGTGGGGAGGTGACTGTTTTGACACATGCCAAAAGAGGGATGGATAGGCTAGAGCCCCAATAACACTAGGGCTCCAGCCTGAGAGGCA

[0038] CCGTGCGGGTGTTTGGAAAATAACGATCAGGATTGGGCGCGGAATGAAAGATATTGATCGTTAATTGACACGGAATGAGCGCCTTACGAT

[0039] CCAATGGCGGGCCCCATTAATGAGGTTGGAACGGCAATGTGGCGTTTCGCTTTCTCTGTAAATGAATCCGAAAGCTGGACGGAACCCCTA

[0040] TTCGAGGGTCAGGACGCGCCAGAAGCCCCGCAGATCAAGGCTATAGCCCGACCAGAGGGACTCCCACGGCCGTACTCACGAAACGAACTT

[0041] CCAATTTCGTTTGTGCGCGAGTTTTAAGCCATCCAGCGCCCTGACCAAACAACGCGTCCGATGATGTCGACGGCGTGCAAGTCTTGCTTA

[0042] GACACGAACATTGTCTGATAGGCCGGGTTAGCACTGATGATCGCGAGGCCGCCATTGATCTGCCGTTGGACGCGCTTGGCGTACAAGTGG

[0043] TCATCCAGGCGGATGACGTAGACAGCTTCGTCACGAACCTCATTGCAACTGTGGTCAATCATCACCGTATCGCCATCGCTCAACAGTGGC

[0044] TCCATTGAGTCACCGCCGATGCGGACGGCCGACATATTCGCGATCTCCAGGCCTTGCTTCCGCAAGCTGTAGCGAGTGAATGCCAACTGA

[0045] GCGAGTATCCGCGCCCCCTCTGTCCAGGAGCCGTGACCAGCGCTCACCCGAGCGTCATACAGAGGGATATAGGCGTAATTTTCGTCTCCC

[0046] TTCTCGCGACTATCTTCCGCAGGTATAGGGGTGCCGAAAGCCAGCCACATAGCGGAAGTCCCTGCGGCCTGGGCGATCTGCTCAAGTCGA

[0047] TCCAAGGTTGGGAAAGTCTCCCCCGACAGATAACTGCGCAGGGTCGCCTCGGACAAGCCGCATTCCTTAGAAAATCCTCGCAGGCTATGC

[0048] GAGGAAATCGCCTGCTTCAGACGGATTTTGAAGTATCCGATTCCATCCTGGGGAATCGGATGCCCAGACAATTCAGCCGGTGCGCCATCC

[0049] GTTTGTTCGTAACTCATTGATTTTCCTAGCATTCATAGTTTTTCCGCGCACAACTGCGAAGAAAAACTAAACGGATGAGTGGAAATCCGT

[0050] TGCTGTGCGAGGATTTTCTCGCTATGTTTATGCCCTAAGTCACGTTAGACACCCCAAAAAAACCACCCTGCACGGTGGTTGAGATGGAAC

[0051] GTAGGCATGAACAGAACCGAAATACCCCGCGACAACAACGTCCGCTGGGAGTGGATCAAATACCAGCTCCGCGCCAGGGGTTCGTCCCTG

[0052] GCCGAGGTGGCCCGCTCCCTCGACGTATCCAGCCCGGCAGTCAAAAACGCCAAGCTGAATCCCTATCCTCGGGTAGAGCGCGCCATCGCC

[0053] GCTGTGCTCGACCTCAGCCCCCTGGTGCTGTGGCCTGAGCGCTGGCTCGACGACGAGAATCCGAAGCGGCAACGGCCCAATCGTTCGGAA

[0054] ACCCTCAAGGCATATGACAGTGTCATATGCCGATCCGGGAAAAATACCAGATACGCTGACCAAGCGCAGCGTAAAGCCGACGCGGGGGCT

[0055] TGAACATGGCCCGCCGCAAAGACGACCGCACCCTGGACATCTTCGATGTTCCGCAGCCAGCTCGCGCTATACCCGGTGAGTGCAACTACG

[0056] CCGCCCAGGTCAGCGAGCTGGTCAGCGAAATTCTGAAGGCTTGCGACCTTGACCGTTACGAGGTCGCCGCCCGCATGTCGCGCCTATCTG

[0057] GCGATGACGTGAGCAAGAACATGCTCGACGCCTGGTCGAGTCCCGCCCGAGCTGATCACAACCTGCCTTTCTACCGCGCCGCCCTCCTGG

[0058] AGGACGTCTGCGCGAGCCACCTGCTGACCAACTGGCTGGTCGATGTGCGGGGCGGCCGCGTGGCCTATGGCCGAGACGCACTGCTCGCCG

[0059] AGCTGGGCCGCCTGGAGCGGACCCGCGAGGAAGCGGGTCGGCAGGCGCGTGAGCTGAAACGAATCCTGGGGGAGCCGTGATGACAAACAC

[0060] CGACCTCAAACCGCTGTTCGACAACCTGCGGAACGCCACCGAGTTCTTCAACTCGGTCGATGGCGACCTCGCCTCGATCTCCAACGGGAC

[0061] CAAACAGATTGCAGCCGAATGGCTGGAAACTGCCGCGCTCGCCCTCGGCGACGCCCTGATCGCCCAGCGCAAAGCTGTCGGGGGCGACCA

[0062] TGAGTGAATGGTTCTCTGCCCAAGAGCTTGCAGGGTTGCCAGGGGTTCCCGGAACCGCGCGAAACGTCAAAGCAATGGCCGAGCGTAAGT

[0063] CGTGGAGTGGACGGCGCCGCGTCGGCACCAAGGCTATCGAGTACCACGTTTCGACCCTGCCGAAGGAAACCCGCGCCGCGCTGCTGAACG

[0064] CCGCCCTGGGCGAGGTGGCCACCAAGGTGGTCCGCCAGGAGACGCAGCTGGCCCTGGTCGAAACCAACCGCCAGCAACTGGTCGCCGATG

[0065] CCCGCCAGGGGGTGCTGCACGCCCTGGACCTGCTGATGGCCCGCACCGGCTACAGCCGGAAGCGCTCCATTACCCTGATGCTGGACATGG

[0066] CGCGCCTCGGCCAGGTCGAGGCGCAGTTGCTCGCCATGCTCAAGATGGCCCGCGATCCTCGTGGCCGCCCGAGCGCGGATGGCCTGCCGA

[0067] GCGTGCGTAGCCTGGAGCGCTTCCTGGACCAGGCCGAGCGTGGCGCCCTGGTGCCGAAGGTCCGCCGCCCGGACATGAGCGTTCCCGACT

[0068] GGGCGCCAGCCTTCATGACGATCTACCAGGGGCCGGAGAAGCGCAGCGCCCGCGCTGCCCATGCACTGCTAGAGAAGCACTGGCAAGGGC

[0069] AAATGCCCAGTCTGGACCAGGTCTATGCGTTCCTGCGCAAGGTGGGCAACGTCAGCCGCGAGGTCGGGCGTATGGGTGAGCATGAAATCA

[0070] AGGCACTGCGCCCGTTCATTCGCCGCGACTTCACCAAGCTACTCCCGACCGACGTCTATTCCTGCGACGGCCACACGTTCGACGCCGAGG

[0071] TCCAGCACCCGATGCACGGCCGGCCCTTCCGGCCGGAAATCACCACCATCATCGACATCCGTACCCGTCGAATTCCGGGATGGTCCACCG

[0072] GCCTGGCCGAGTCGGCCCTGGTGGTTGTCGATGCCCTGCGCGACGCCTGCACCAAGGGCGGCATTCCGGCCATCTTCTACGTGGACAACG

[0073] GCTCGGGCTACATCAACCACATGATGCGCGACGAGGCGGTCGGCCTTATGGGCCGCCTGGGCATCGATATGAAGAACAGCCTGCCCTACA

[0074] ACAGCCAGGCGCGGGGCGTGATTGAGCGCGTCCACCAGAGCCTGTGGATTCGGGCCGCCAAGGAACTGCCCGGCTACATCGGCGCCGACA

[0075] TGGACCGCCAGGCCAAGCTGGCCACCTTCAAGCTGACGCGCCGTGCCATCGCCAAGGGCGGCACCATGCCGCTGATGTCTTGGGAATCTT

[0076] TTGTCGCGTTCTGCGAACAGCAGATTGCCGAGTACAACGACCGGCCGCACAGCAGCTTGCCGCGCATCGTTGACCCGAACACCGGCCGCC

[0077] GCCGGCACATGACCCCCAACGAGGCCTGGGCGCTGCACGAAGCGGAGGGTTTCAGCCCGATGCGCGTCACCGACGACGAGGCCCGGCCGC

[0078] TGTTCCGGCCCCAGGTGCTGCGCACCGTCCGCCGCTGCGAACTGGAGTTCATCGGCAACCGCTACTTCGCCCGCGAGCTGGAGGAATTCC

[0079] ACGGCGACCAGGTGGCCGTGGGCTACGACATCCACGACGCCAGCAAGGTGTGGGTCTACGACGGCGAGGGGCGCTTCCTCTGCACCGCAG

[0080] AGCTGAACGGCAACAGCCGCGACTACATGCCAGCTTCGTATGTCGAGCGTGCCCGCGAGAAACGCGCAGAGGCCCGCGAGAAGCGCGCCC

[0081] TGGCTCACCTCGACGAGATTCGCGCCGAGCGTGACGGCGGGTATGCCCTGGAAATGGATGCGCCGCTGTCCATCCCCGGCCTCGGCACAA

[0082] TCACCCCCGAGCAACTCCGGTCGCGCAGCGCCGCGACCCTTGAAGTGCAGGCCGAGCGGATCGACGAACCGCGCCCGGCCGCAGCCGCCA

[0083] CCCAAGCCACCACCGCCCAGGTCTTCACCCTGCCGACCGCTCCCGCCCAGCGCTACCGGCAGTGGTGCGAGCTGGCCGAGCGGCAGCGCT

[0084] CCGGCCTGCCCATCGAGCCGGACGCCGCCCAGTGGTTCGAGGTTTACCCCAAATCCAAAGAATTCGCCGCCCAGCAGCGGCAAGCATGAA

[0085] AGGAGCTGTATTCATGACCACCCCGAAAACCACCCAACTGGCCAATGGCATGGCCGACATCGCCAACATCGCCCTTTGCGATATCGCCCT

[0086] GGAGAAAGCGCTGTCGCGTACCTCCACGTTGCCCGGCCTGGTCTGCTTCTACGGCCCGTCCGGCTTCGGCAAATCCGTTTCGGCCGCCTG

[0087] GGTCGCCAATCGCCGCCGCGCCTACTACGTCCAGGCCAAAAGCGTCTGGACCCGCAAGCACACGCTGAAGTCGATCCTGGGTGAAATGGG

[0088] CATCAAGCCGGCCGGGACCATCCCGGAAATGGCCGACCAGATCGCCGAGGAACTGGCCGCCAGCGGCCGCCCGCTGATCATCGACGAAAT

[0089] GGACCACCTGGTCGCCGCTGGCCAGGTCGAGCTGATTCGTGATCTGTACGAGTCCAGCCAAGCCTCCATCTTGCTGATCGGCGAGGAAAT

[0090] GCTGCCGACCAAGCTCAAGAAGTTCGAACGCTTCCATGGCCGCGTGCTGAGCTGGGTTCCGGCCCAGCCAGTGTCCCTGGAGGACGCCCG

[0091] CAACCTGGCGCCGGTCTACAGCCCCGGAGTGGCTATCGCTGACGATCTGCTCGCGCACCTGGTGAAGAAGTCCCTGGGCTCTGTCCGTCG

[0092] CGTCGCGGTGAACTTGGAGCAGTTGGCCGAGGCCGCCACCGTTCAGGGCCGGCGCGAGCTGGAACTGGCCGACCTCCAGCGCCTGAACCT

[0093] GGAGCTGTACACCGGTGCGGCCCCGAGCCCGAGGACTTCGAAATGAGCCTCGGCAAGAACCCGGCTCATCTCTGCATGGTCGGGGGCAAG

[0094] AGCCCCCGCCAGCAGATGTGGGAAGTCATCCGGGCCAACCGCGAAGAGTTCACCGTCTACCGCGTGGCGCGCCGCTCCAACCAACACGAC

[0095] AAGACCGTCGAGAAGTATGTCGCCTGCCTGCGCCTGGGCGGCTACGTCGAGGCGATCCGCGGATTCAAGCGCGGCGAAGAGGTCGTTTTC

[0096] CGGTTGATCCGCGACAACGGCGTCGAGGCACCGAACCTGAATGCCGATGGCAAGCCATCCCAGCAGGGTTACACCACCGAGGCGGTCTGG

[0097] CGGACGTTGCGAATCCTCGGCCCATCGACCCCGGAACAGATCGCCGCATCGGTGGCGGCCTCGGGCACGCCCGTGTCGCCCAGCACCGTT

[0098] CAGCGCTACTTCATCGACCTCCAAAACGCCGGATACCTGACCCGCAACGGCCGCCACTACGCCCTCAAGCCGGGCCGCTACACCGGCCCT

[0099] CGGCCGCCCATCGTCCAGCGCGAGACGCGCCGCCAGGTCTACGACCCGAACCTTGATCAGGTCATGTGGAGTTCGCACGGCGAGTACCAG

[0100] CACAACCGGAGTCGCTCCAAGAAATCAGAACAGGAGGTGCCGCCATGCGCCGAACACTGATCCCCATCGGCATCTTCCTGGCCCTCGGCC

[0101] TGCTGCTGATCCTGGCCGGTGATGCCCTGATGCTTGGCCGCCGCCTCATTGCCTGGCAGTGGGGGTGCTAATGGACCGCGCAATCGATCT

[0102] GTCGGCCTGGGGCGAGCGTCCGCCCGTCTTCGTCCAGCTGCTGGCCGCCGAGGTGGCCCGCAGCAGCCAGACGAAAGCCGGCGAGGCAAT

[0103] CGGCATGAGCCGTTCGACCGTCAGCACCATCCTCGCCAACCGCTACCCGTCGCCCTCGACGATCCGCGTCGAGCGTCGTGTCCTGGCCGC

[0104] GCTGAGCCGCATTGAGTGCCCGGCCCTGGGCGAGGCGGTGACCTCGGTCGAGTGTAGCGAGTACCTCCAGCGGCCGGCGCCGCTGAACAA

[0105] CCCCGTCGCGATGCGCTGCTGGAAAGCGTGCCGCGCTTGTCCACGCAACCCGCATACCGCCCCCATGAAACGAGAGGAACAAGGCCATGA

[0106] GAACCGCATTGCCCTTGAAAGTGTTGACGCCTGACTTGGCCCAGAGCCTGCGCACCTTCAACGATGCCGCCCGGCTGCTCCAGCGCATGG

[0107] GGGTTCGCCTTCATCGCCTGGAGCCGACAGAGGGGCGCGTGACCATCGCCGCAGACGACGCTCGCCAGCTCCTGGAGAAAGGCTACCTGA

[0108] TGGGCTTCCAGCGCGACGCCTCGGCCGGCAGCACCCGTTACATCACCCGCTTCCAGGGCATCACCCTGGCCTGGAGCGAACCGATCAGCT

[0109] ACCGCGACTTCGCCGGCAGCAACCCCGTAATTCACTGAAAAGGACTACCCCATGAACGATGTACATATCCCCGAGGGCTACCGGATGGAC

[0110] GGGCAAAAGCGCCTGATCCCGGAGGCCACCATCAAGCCCGTTGACCTGGCCCGCGACCAGTTGGTTATGGAGCTGGTGGGCAAGGCCAAG

[0111] GCAGTCCAGAGCGAGCTGGCCCGTTTCAAGGCTTCCGCGTTCGGCGACATCGACGCTTTCGTCGATCTGTCGGTCGAGCAGTACGGCACC

[0112] AAGCTGGGAGGCAAGAAAGGGAACGTCTCCCTCCTGAGCTTCGATGGCCGCTACATGATCAAGAAAGCGATCCAGGAAACCATCCAGTTC

[0113] GACGAGCGTATCCAGGCCGCCCGCGAGCTGATCGAGGAATGCCTGCGCGACTGGACCCAGGGCGCCAGGCCCGAGGTGGTCACCCTGGTA

[0114] ACCAACGCTTTCCGCACTGACACGAAGGGCGAAATCCGTACCGCCCGAGTGCTCGCCCTGCGCCGCCTGGACATCCAAGACGAGCGCTGG

[0115] CTGCGAGCCATGAATGCGATTGGCGACGCCTGCCAGGCGGTGGGTTCCAAGGCCTACATCCGCGTCTATGAACGGGTCGGCGACAGCGAC

[0116] CAGTACCAGCAGATCAGCCTCGACATGGCGGGGGTGTGATATGGCCGACACCATCGCTTTCTGCTGGGCCTCTGGCTTGATCCAGTTCGG

[0117] TGACCAAGTGCCCGACGGTGCAATCGAGATCGCCCGTGGGGACGACCAGGTCGTTCGCGAAATCATCGAAGTCACCTCGCGCCACGCCTA

[0118] CGACGGCAAGTCGCTCCTGGTGCCCGGCATACCCGAGGCAAAAAACCAGGCGGAAGGCATTGACGCCCTCGAAAGGTTCGTTCGCTGGAT

[0119] CGCCTCGCACAACAGCGAGTCGTTCCAGGTTTTCATGGGAGACGTGTGACATGCCTACCAACGTGGAGCTTGTAACCCGCTTGATGGAGT

[0120] ACCCCAAAAGCGGACCGCTGATGCAAGCAATGGTCCTCCAAGCCTTGGATCAGTTCGCTTGCATGGTACTGGCTTCGGAGCCGGGGTCGC

[0121] TCGAAAGCCCCATGGTTTCCGAGGCGTCCTGGCGCGCCTGCGCTGAGGAAATCCGGGAAAGCCTATCTAGCCACATGCGTCGGGAGGCGT

[0122] GACATGACCAAGACGTTCGCCATGTGCCGCATCGACGGCCTGATCGAGCTGCGGGAGGAACACCCAGGCGAGGGCTACTTCGCCCTTGCC

[0123] GTGGGCGACTTGGCCAGCGTGCGGGCGGCGGTCTTTGCAACCGCTGAGCCGCACCAGGTCGGCAAGAAAGTCGCCCGGCGCGTGCCGGGT

[0124] GTGAGCCCCGACGCCACCGACCGCGAAAACCTGGGCTCCATCGCCCGCTACATCCAGACCCTGGGCCAGCAGGATCGGCCCGGCTTCCGT

[0125] GCGCTGGGGGTGTGAAATGCAGCAGTCCAACCCCTTCAATCATCCCGGACAGAGCTACGGCGCCGTAGACGTCGATAGCCGTCTCCGCGC

[0126] CGTTGCCGGCTTCGACCTGGAGCAATGCCGCGCCGCGCTCGCGGTCACCGGCCTCCAGAAGATCGTCGAGCAGAAAATTCGCACCCGCAT

[0127] CCGCCAGCTGGAAAAGCAGGTATCCGCACAGAAGGAGGCATAACCATGGCCGTATACACCATCACCCTCAGCGACACCGAAGGCGGAATA

[0128] AATTTCTCCATGCAAGGCCCTCCGCTGCACGACTCCGAAGCATCGAAGGTCGCCTATGCCCTTATGCAAGCGACGATGTCCCTCGGCCAG

[0129] GAACTCGCAAAGCAGAACGGAGCTGGTAACGGCGTTTCCTGCGCCTGCGACGAGTGCCTGGCGCGCCGCGCTCGCGGCGAAGAACCGCAA

[0130] CAGGAAATCCACTCTACCAAGGCCAAGAGCCGCACCGTCCATTGAGCGAAACCGCCCCGGCCTGGCCGGGGCGGTCTGCCGGACGTGGTG

[0131] GTCCGGTACTGACGAGCAGCCGAGGAATACATGGAACCCAACAAAGCCCTGGAAAAAATCAAGAAACTGCTTCGCCTAGCAGGCAGCAGC

[0132] AACCCACACGAAGCGGCGGCCGCCATGCGCCAGGCGCGGGCGATGATGGAGAAACACGGCATTGGCGAGAGCGATATTGCTCTGGCTGAC

[0133] GTCACCGAACACGGCACCGGTAGCGGCTCCAAACTGAAGCCCGCGCAGTGGGAAGCCAATCTCGGCGGCACCGTCGCGAAGGCATACTCC

[0134] TGCAAGCTGTTCTTCGCGGCCGGCATCGGCCAATGGCGCTTCGTGGGCGAAATGGCCGAGGTGGCCAGCTACACCATGGCGATCCTGCTG

[0135] CGCCAGGTGCGCAAGGCCCGCCGCGACTACATCGCCACCGAGCTGAAGCGCTGCAAGGCCTCCACCAAAACCAAGCGTGGCGACCTGTTC

[0136] TGTGACGCCTGGGTGTGGTCGGTGCGTAAGCAGGTCAATCAGTTCGCCGGGAGTGCGCAGCCGTCGCTGGCAGCCGAGGCGTACATGGTG

[0137] AAGCACCACCCCGACCTGACCACCGGGCAATGCACCGACCGCAGCAAGAAAGGCCCGCTCAGCAACCGCGACCTGGGCGACGTAGCCAAA

[0138] GGCATCGCTGCGGCTGATGGCGTCCAGCTCAACCATGGTGTCGGCGGATCGGCTCCGCTGGCCTTGACCTGAGCGCGTCCGGCGATGACG

[0139] ACCGAAACCAAAGCCGAACGTCGGCGCCGGATGGCGCGGGAACGCCAACGGGCGAAGCGCGAGCGCGATGCCCTGCGCCGCGCTGCGCTG

[0140] GGCGGCCGCCGCTTCAACATGGACATGTACCAGGGAACGGCTGATGCACTCGATCTGATCTGCGCGGCCGGGGGCTTTGCCGAGCCGGCC

[0141] GAGGCGGTCACTTTGCTCCTACACAACGTTGCCGAAATTGCTGAGCGTGACGCGTCACGTTTTGCCGAATTGATCCGAAAGAGAAGCCAT

[0142] CCAGGGAGGACCAAGCGATGAGCCTACGCGCCGTCAATCTCGCAAAAATCCACATCGCCAAGGCCCAGCTCGGCATGGACGATGACACCT

[0143] ATCGCGCATTGCTCGCTCGCGTCGCGGGCGTGCGCTCGGCCAAGGACTTAGGGCCGCGCCAAGTGGGTAAGGTGCTGGCCGAGTTCCAGC

[0144] GCCTGGGCTGGAAACCGAAGAGCAACCGGCAAGGCCGGGCGACGCCAAAAGTGCCGCAAAACCGGCAAACCGTGCTGCGCAAAATCACCG

[0145] CGCTCCTGGCCAGCGCCCATCGCCCCTGGAGCTACGCCGACCACATGGCCCGGCGCATGTTCCAGGTCGAGCGGGTCGAGTGGCTGGACG

[0146] ACAGCCAGCTCTACCGGCTGATGCAGGCGCTTATCATTGACAGGAGCCGCCATGGCCAGGTCTGAGGTGGATCTTCGGGAGGTCCAGGAC

[0147] ATGCTGCCGGATACCGTCCGCGACATGGCCGGACGCATCGGCCTGCCGGCCACCCTGGTGGTGGTCGAGCAGCTCGGCGGCACCTCCTGG

[0148] CGGATAGCCGAGGGCCGGGCGCGTAGGGGCGAAGCGCGCCGGGCTGCGCTGGCCGAGCTGGTGGGCAGCGAGATCGAGGAGCAGCTCCAC

[0149] ACGCACTATCGGGGCGAAGAAATTTACGTGGCTCGCTGCCACAAGGCACTGGTACGGTGGCGCGACCTGGAGATCGTCGAGCGCTTCGAA

[0150] CAGGGCTTGCGTGATGGGCAAACCGCCCGTAGCCTGCTCAGCGATCTTGCCCGCCAGTACAACCTGTCCGACCGCTGGATATGGGAGATT

[0151] GTCAACCGGCCGAGCGAGCCGGCACCGCAGCAATCCACCCTGTTCCACTAAGCCGGGGCGCAACGCCCCGGCCGGCGTCTCCGCGCCGAT

[0152] CCTGTCTCAGCCGTTGAACCCCTTCCGCTAATCCCGCGTCGCACTCGCCGCCACGATGGCGGCATGAGCACATCTAGCCCCCCAACGTCT

[0153] CTACGCAGCCCCCGCGACTACGCCGCCGCCATCCTGGCCGAGCCCAGCCGCGAGCGTCGTAACGCTCTGCTGGCAGCCTGCCCGGTCAAC

[0154] TGGCAGCCGCTTGTTCGGGCGCACGTTGAGGACGCTTTCGCGAAGGTCAAGGCGTATCGCCAGATGATGGACCACCGCGCCGAGTCGATC

[0155] CGGCGCGGCCCGCCTCCTGCTCCCCGCGTCACCGACACCGATTTCCGCATATCCAACTACACCAAGTCCGCCCCGGAGGTAGGCAATGCG

[0156] CACCTATCCGCAATTCGGGCAGCGCTCGCATCGGAAGCACCAAATGCCTGATCCCGCATCCACCTCGGCCGGCAGCGCCGCGCTGCTGAA

[0157] AATGTTCGGCATCCACATCAGCGCGGGCGCCCTGGCCGCCGCCCTGGGCTTCCTCGTCCTGTGGCCCCGGACCATGAAAGAGGGGTTCGC

[0158] CCGGCTGTTCTGCACCATCGTCGCGTCCAGCGTCTTCGGCCCGATCCTGGTGGTTTACCTGCACTCCAACCGCCCCGAGCTGTTCGAGTC

[0159] CGCCCAGGTGGTGGCCGGGCTCTACCAACTGGAACCAGCGGTCGGCCTGCTGTTCGTTTCCGCTCCGCTCCTGGTGATTGCCGGTCTGCC

[0160] TGCCTGGTGGCTGATCGGTGCAGCCCTGCGCCTGTTTGAGCGGGACGGCGATTCATGGCTGGGCGCGTTCGCCCAGTGGGTAAAACGCAA

[0161] ACTGGAGAACAACTGATGGCCCTTCAACCTCGCGGCATCCGCAACAACAACCCCGGCAACATCGTTTGGTCGGCACGCAACAACTGGCAG

[0162] GGCCAGCTCCCGCACAACCCGAAGATCGAGCCCCGATTCGCCCGCTTCGACACCGCGCATAACGGCATCCGCGCCCTGGCAAAGCTGCTG

[0163] CTGAACTATCGCAAGGTCCACGGCCTGCGCACCGTCGAATCGCTGATCGCACGCTGGGCGCCATCCAACGAGAACGACACACGCGCTTAT

[0164] GCCACGGCCGTGGCCCGAGCCATGGGGGTTCCGCCGCAGGCCGGCCTGCACATGGACCAGGACACACTGGCCGCCCTGGTGACCGCGATC

[0165] ATTCGGCACGAAAACGGGCAGCAGCCCTACAGCGCCGAGCAGATCGCCCAGGCCGTGCGGGAGGTGCTGTGATGCAGCGCCCCAGCGGAA

[0166] TCAGCCTCAGCGATCTGTTCGCGATCTGCCGTGAAGACCCCGGCAATCGCTGGCTCTGGATACGCCTCTATCTCCGCGACCTGCTGGCCC

[0167] GCGTCGTGATTCTGGTCGTCATGGCAATTGGTGCCGCAGGCCTCGCCTATGGCCTGGGCGGGGCGTTCGCCTACGGCTTCATGCAGACCG

[0168] TTGCGTCCTACCAGGTCCAGCTCAGCGTCGAGAAATCGCCATGACCTGGCGCGTCGACCTGGTTGTCTTTGTGCTCCTGGTGATGGTCTG

[0169] GACGGCCGGCTGGTGGGGCGGTCGCGAGGCTGGCCTGGCCGATGGGCGCGCCGCCTGCGCCGACGCACAGACCCGCGCATATCGCGACGT

[0170] CCTGGAGCAATCGGCGGCACAACTGAAGACGGTCCAGGACACCAGCGCGGCTCTTTTCCAGCGCCTGGCCCAGCGGGCCGACAGCGACCA

[0171] ACAAACTACTCGGGAGCTTCGCCATGCCCTGGCCGAAACCGCTGCTGATCGCGCTGCCTGCCGCTTTCCTGCTGGCGTCATGCAGCAGCT

[0172] CGAAACCGCCCGTCAACGTGCCGCCCAGGCCACTACCGGCGGCCTTGGCTCAACCGTGCCCGACCCCGGTGGCGGTGACTGATGACAGCC

[0173] CCGATGCCACCGCAATTGCCCTCAAACAACTCTACGACCAATACGGCGTTTGCGCCGGCCTGCACTGGGACACCGTGCGGCACCTTCAAA

[0174] AGGACTGATCCGATGACCGAAAAGAAAGCCTCTCCCGAGTTCGAACTGCTGCAACGCATCGACGGCCGCCTGGAGCGCTTCGAAGACCGA

[0175] TTCCCGCAGATCGAACGTAAGGCCGTGCTGTACGGCTCGGCGGCCGGCGCGCTGGCGGGTGGCCTGGTTGCCTGCGGCCTGCTCGCGGCG

[0176] CGTATCAAGCTCGGTATCTGAGGTAGTCCATGGCGCACCCGAAGGAAACCCGCGACGCCCTGCGCCGCGCCTACGTCCTCGACCGCCAGT

[0177] CCCTGGAGGTCGCGGCCGCCATGTTCGGCGTCTCCTACGGCACCGCCCGCCGCTGGAAACAGCAGGCGGAAGCCGAGGGGGACGACTGGG

[0178] ACAAGGCGCAATCGGCGCAGTTGCTGGCCGGTGGCGGACTGGAGGACGTGGCGCGCCAGGTGCTGGCCGGCCTGGTGACCCAGTTCCAGG

[0179] CCACCATGGAGGCGATCCAGGTGGACAGCGCGATCACGCCAGCGGTCAAGGTGCAGATGCTCGCCAGCCTGGCCGACGCCTACAACAAGT

[0180] CGATCAGCGCATCGAAGCGGGTACTACCGGAAACCTCCAGCCTGGCCACCGCAATGGAAGTCATCCAGCGCCTGGCGGCCTTCATCCGTG

[0181] AACAGTTCCCGCAGCACGTCCAGGCCTTCGCCGAGATTCTGGAGCCGTTCGGGGAAGTAATCGCCAAGGGGCTGAAATGAACGCCGAGGA

[0182] AAAAGAGTTCCTACGGGAACTATCCGCCATGGCGCAGCAGCTGCGCCGCGACATCGAGGCGCAACAGGTCGGCCTCGATAGCTCCCCGGA

[0183] AGCTCGGGCCGAGCGGCGCCGTCGCGTACTGGTAGATCGCGATTTCGAGTTCTTCGCGTATACCTACTTCCCGCACCACATCCGGCCGCC

[0184] TGCCTCGTTGTTCCATGCGCATTTCTTCAAGCGCTTTCCCCAGCTCATCAGTAGCTCCAGCGGCCTCAAGGAATGGTGGGTAGCTCCGCG

[0185] TGGCGAGGCGAAGTCCTCACTGCTGACCAAGGTCGGGCCGTGCTATGTCGTTGTCCAGGGGCTGCTCCAGCGACCGGAGATTCGGGCCGA

[0186] ACTGGGCATGACCGGCCCGGCGCCGTACTTCGTCGACTACATCACGCTCCTAGGCGCCGAAACGCGCTTGCCCACCAAGCTCCTGGAAGT

[0187] AGTCAAGACCGAGCTACTGGTAAACGCCTCCCTGTCCCTGGACTTCCCCGAAGTCTGCGGCAAGGGAAGCGTCTGGAAGATCGGCGAGTT

[0188] TGTTTCCCTCTCCGGGGTCAAGCTGGAGGCCTTCGGCGCTGAACAGGCGATCCGGGGCACATTCCACGGCGCGAGCCGTCCCAAGCTGCT

[0189] CCTGGGCGATGACCTGATCACCGACAAAGAGGCCAAGTCCCCGACCGAGCGCAACAACCGATGGGACTGGCTGGAAAAGGCTATCGACTA

[0190] CCTCGGCCCACCGGATGGCTCCGTCAAATATCTAGGCGTGGGCACAGTGCTGAATAAGGACGATCCGATCAGCCGAGCCAAACGCACGGT

[0191] CGGGCACCTGGTCCACCACTTCCGCGCTATCGAGACGTTCCCCACCCACATGGACCTATGGGCACACTGCGAAGAGGTGATGCTCAACGA

[0192] CGATAAGCCCGTGATGGAGCAATACGCCGAGCGCGGTAGCGTCGCGCCCGATAGCGCTCTGCCGTCGTTTCAGTTCTACCAGGACAACCG

[0193] CGAGCAGATGGAGCTGGGGGCCGTCACCAGTTGGCCAGGTGTCCGGTCGCTCTACTGGCTGATGCGTCAGCGGGCGAAGAACAAAGCCGC

[0194] GTTCGCGACCGAGCTGCAAGGCGACCCGCGATCCGATGAAGACAAGACGTTTACCAACCCGCGCTTTTGGGTCATGCGCTCCGGCCGTTG

[0195] GCAGATGTTTGGCGCCTGCGACCCTTCTGTCGGGGCGAGCGCACAGTCCGACCCGTCGGCAATCCTCGTGGGGGGCTGGGACACCGAGAA

[0196] ACAGGTACTCAACGTCATAGAGGCGGCCATCAAGCGGCGCGTCCCCTCGAAACTGGAAGCTGACCTGATCGCAGCGCAGAGGGAGTACCA

[0197] GATGCGCGCTATCGGTTTCGAGAACAACGGGGCATTCGAAATCCAGCGGCAGAACATCGCCAAGGCTGCGCTGATGCAGCGCGTAGCGCT

[0198] CCCTCTGGTCGGTGTTACCAGCATGGCGGACCAGTCCGTTCGAATCGATGCGATGGAGCCGTTCATCAACGACGCATTTGCGCCACGGAT

[0199] TCTGTTTTCGCCTGGACTGGTCGCCCTGCTCGACGAGCTGGACAGTTGGCCAGAACCGCAGACCGGGCACCACTACGACGGCCTTTGCGC

[0200] CCTGTCGATCCTCTGGATGATCGCTAGCACCCGCGCTGGTTCCTATGAATTCACCCCGGTGCCTGGCCGTCGCAGCTCTGCGGATTCCAG

[0201] TGAGTTCAAAGACTCTTTTGACATAGGTGGCCGCATGGGCGGCGACTGGTAGGAGACGCAACGATGGCCACCATCGTGGATATTTACGGC

[0202] AACCCCCTGCGAACCCAGCAGCTGCGCAAGCAGCAGACCGCGCACCTGACGGGCCTGGCCAAAGAGTTCGCCAACCACCCGGCCAAGGGG

[0203] CTGACCCCAGCCAAGCTGGCTCACATCCTGATCGAGGCCGAGCAGGGGCACCTCCAGGCTCAGGCCGAGCTGTTCATGGACATGGAGGAA

[0204] CGCGACGCCCACCTGTTCGCCGAAATGAGCAAGCGCAAGCGCGCTGTCCTCGGCCTGGACTGGACCATCGAGCCGCCGCGCAACGCCTCG

[0205] GCCGCAGAGAAGGCCGACGCGGAGTATCTCCACGAGCTGCTGCTCGACCTGGAGGGCATTGAAGACCTCATGCTCGATTGCATGGACGGC

[0206] GTCGGCCACGGCTATAGCGCTATCGAGCTGGACTGGTCGCTCCAGGGGCGGGAGTGGTTGCCGCAGGCCTTCGACCACCGGCCGCAAAGC

[0207] TGGTTTCAACTGAACCCGGACGACCAGGACGAGCTGCGCTTGCGCGATAACAGCATCGCGGGCGAGGTACTCCAGCCGTTCGGCTGGATC

[0208] ATGCACAAGCCGCGTTCGCGCTCGGGATACGTGGCGCGTAGCGGGCTGTTCCGCGTGCTGGCCTGGCCGTACCTGTTCAAGCACTACTCC

[0209] ACGGCCGACCTGGCGGAAATGCTCGAAATCTACGGTCTGCCGATCCGGCTCGGGAAGTACCCGCCCGGCACGCCGGACGAAGAGAAGGTG

[0210] ACCCTGCTGCGGGCCGTGACCGGCCTCGGCCATGCCGCAGCAGGCATCATCCCCGAGAGTATGTCCATCGAGTTCCAGGAAGCGTCGAAA

[0211] GGCTCGGCCGAGCCGTTCATGGCCATGATGCGCTGGTGCGATGACTCAATGTCGAAGGCCATCCTGGGCGGCACGCTCACCAGCCAGACC

[0212] AGCGAGTCGGGCGGTGGTGCCTATGCCCTGGGGCAGGTCCATAACGAGGTGCGCCATGACCTTCTGGCGGCGGATGCCCGACAGCTCGCC

[0213] GCCACGCTGAGCCGAGACCTACTCTGGCCCCTCCTGGTCCTCAACCGCTCCGGCAACCTCGACGCGCGCCGCGCCCCCCGCCTGGTGTTC

[0214] GACCTCAAGGACCGGGCCGACCTGGCCGCCATGGCAACGTCATTACCGCCCCTGGTCAAGCTGGGCGTCCAGGTGCCGGTCAACTGGGTC

[0215] CAGGAGCAGCTGGGAATCCCGCTGCCAGCCAATGGCGAGGCGGTCCTGGGCGATCAGACCGGCGCAGGCATCGCCCAACTGAGCCGGCGC

[0216] CCTGGTCCTCGCATCGCTGCGCTGGCCCAGGTGATTGGACCACGCTACCGCGATCAGGAAGCGCTGGACCAGGTGCTGGCCAGCCTGCCG

[0217] GCCCAGGACATGCAAAACCAGGCCGATAGCCTGGTCGCGCCGCTCCTGGATGTGATCAGCCGCGGAGGTAGCGAGGCCGAGCTGCTCGGC

[0218] GCCCTGGCCGAGGCATTCCCGGATATGGACGACAGCGCCCTGGCGGATGCCCTCCATCGGTTGCTGTTCGTGGCCGACACCTGGGGCCGG

[0219] CTCAACGGAACGCTGGATCGGATCGACTGATGGCAACGCCAACCGAGGCCGATCTGCGGGCCATCTTCGCCATGCGGCCGGAGGCCGCCA

[0220] TCGAGTACCTGGAGCGCAAGGGCTTCTCGATCACCTGGAACTGGCACGATGTTGACGCAGCCACCCATGCCCGAGCGCTGACGGTGGCCA

[0221] AGGCGGCACGCCTGGACGTGCTCCAGGACATCCGCGACGCCCTGGTCGATAACCTGGAGCGTGGCGGAACGCTGCGCGACTTCCAGCGCA

[0222] ACCTGCGGCCGATCCTGGAGGCCAAGGGCTGGTGGGGACGCCAGATTGTGGTTGCGCCGGACGGCGGCGCCGAGGTCGCCCAGCTCGGCA

[0223] GCCCGCGCCGGCTGGAAACGATCTACCAGACCAACATGCAGTCGGCCTACATGGCCGGGCGCTATGCCGCCGCATACGAGGCCAGGGAAA

[0224] CTCACCCTTACTGGATGTATGTAGCCGTTATGGACAGCGTCACTCGGCCCAGCCATGCGGCGCTACATGGCAAGGTGTTCCGCTGGGACG

[0225] ACCCGATCTGGCAGCACATCATGCCGCCGAATGGCTACAACTGCCGGTGCCGAATCGTTCCGTTGACGGCGGCCGCTGTGCGTCGCCGTG

[0226] GTCTGACGGTCGAATCCAGCGTCGGCAAGACGGGCCAGGTGACCGTCGAGACGGGCGTAGACAAGCGGACAGGGGAGATTCGGGAGCAGA

[0227] CCTTGACCACCCTGGAGACGACCGACCGGGCCGGTCGGAAGATCCAATTTCGCCCCGATGCCGGGTTCGACGGCAGCCCGATACAGAGCG

[0228] CGCTGATGGACCAGGTGCTGTACAACAAGGCCGAGCGCACCCTGGGAGCGCCTGCCGCCCTCGGCGAGGTCCAGGACGTGCTCCTGGACC

[0229] CGGTACGCCAGCGCGCCTGGCAGGCGTTTGTGGACCGCTCCACGTCGCCCCAGGGACAGACGATGTCGGTCGGCGTCCTCGATCCGACCG

[0230] ACATCACCTATGCGGCTGCCCAGGGTGCCCAGCTCCAGGCTGGCGTGGTATCGGCCAGCGACACCGTGATCCGTAACAGCCCGGTCGCTC

[0231] GCGAGCAGCTGGCGAACCTGCCGCAGCGCCTGGCCCAGCCCGCGATGGTGCTGTGGGAGCGTGGCAGCGAGTCCTTGGTCTATGTCGTCC

[0232] AGGACGGCGACTCGACCCTGGCGGTTCGGCTGCGCGGTGGCATCTATGGGCCGGGCCAAATGGAGAACATCAGCCAGGTGACAGAGGTGA

[0233] CGATGGAAAGCATCGACGACGGGCTCGCCCTGGGCCGCTACAGGAGGGTTCGCTAATGGCCAATCGCATCGAGCTGGAGCTGGTGGACCG

[0234] CGAGGTCCAGGAGCGCCTGGCGGCACTCTACGCGGCTGTAACCGACACCCTGCCGCTGATGCGCGGCATTGCTGCCGAGCTGCTGGCCGA

[0235] GACGGAGTTTGCATTCATGGACGAGGGGCCGGGATGGCCTCAGTTGAGCCCCGTTACCGTTGCAGCGCGTGCGGCGAAGGGGCGCGGCGC

[0236] GCATCCAATTCTCCAGGTCACCAACGCCCTGGCGCGCTCGATCACCACCCGCGCCGACCGTGACCAGGCGCAGATCGGCTCTAATCTGAG

[0237] CTACGCAGCTATCCAGCAGTTGGGCGGTCAGGCTGGGCGAGGCCGCAAGGTGACAATCCCGGCGCGCCCGTATCTGCCGGTCCTCAGAAA

[0238] CGGCCAGCTCAAGCCAAGCGCCCGCGATGCGGTCCTGGACGTCCTCCTGGCTGTTCTGTCCCAGGGTCGCTAGAGGGAGTGCATTCGGAT

[0239] AAGCGGCACATTGTGCCTATTGTGAATTAGGCACAATGTGCCTAATATGGCGTCATGCCAGCCACAACGGCGAGGCGCCAAGAAGGATCG

[0240] AACCCATGAGCACCATTTCAGATCGCATCGTCGCCCGCTCGGTTATTGATGCGGCTCGATTCATCCAGTCCTGGGAGGATGCGGACCCCG

[0241] ACAACCTGACTGAAAGCCAGGTTCTGGCCGCTTCTGGTTTCGCCGCCAGGCTCCATGAGGGGCTCCAGGCTACCGTCCTGCAACGGCTGG

[0242] TGGACGAGTCCAATCATGAAGAGTATCGCGAGTTCAAGGCATGGGAAGAGGCGCTGCTCAACGCAGATGGGCGGGTCGCGAGCAGCCCGT

[0243] TTGCCGATTGGGGATGGTGGTATCGCATCGCCAACGTGATGCTGGCCACTGCCTCGCAGAACGTAGGTGTCACCTGGGGAAGCCGCGTCC

[0244] ATGGGCGTTTAATGGCTATTTTTCAGGACAAGTTCAAGCAGCGGTATGAGGAACAGGCATGAGCCGACCGACAGTCGTTACGGTGACGGA

[0245] AGTTTCCTGGAACCCAGGCAGTTATGAAGTCAACGTAGAGCAGAATGGCAAGATGGTCGTCGGCCGGACCAGCGCTGGCCATGATCCTGG

[0246] CGCGGCTGCGGCGAAGGCCATGCAAATGGCCATGGAGTGGGGGAATCCGAACTACGTCATTCTCGGTAGCAACAAGGTTCTTGCGTTCAT

[0247] ACCGGAGCAACTGCGGGTGAAAATGTGAAACCTGACGCATCCAGCCACAATCCAGACCCGCGCTACCTGCGCGGGCTGCTCAAGAAAGCC

[0248] GGTATCAGCCAGCGGCGCGCAGCCGAGCTGCTCGGCCTCAGTGACAGGGTGATGCGCTATTACCTGAGCGAGGACGTCAAGGAGGGCTAC

[0249] CGTCCCGCGCCGTATACCGTCCAGTTCGCCCTGGAGTGCCTGGCGAGCGACCCGCCATCTGCGTGATCACCTGATCCGCCCGCAAACGCG

[0250] CTACACGCGCCGAAACGGGGTTAGCCGCTACCTCGCATCAGAGTCGGTGCGTTAACCCCGTTAGAACCCCGTTAGAAATCGTTCCATCGC

[0251] CATCCGCGTGCCAGGGCTTGGCCAGAAGATGGCGCCGGACGGTTTCCGCAGTCGTTGAACCCCTTCACGTAACCGCCGTGCTCGACCGTC

[0252] GCCACCATTGGCGGCATGAAGAAGAACCGCCTACACGTTGCCATCGCCGCCTGCTCTTTCCAGCTCCCCAAGCTGGAGGACGGCAGCGCC

[0253] TGGATTCAAGTTACGCCTGCCGGTGAGTTCTGGCCCATGGATGGGCGCCCTATGGATGTGCCGGGCTGGCGGATCGATGCCGCCAGCGCC

[0254] GCCGCAGTGATCGAGCGCGCACGGTCGCGCAAGACTCCGCCCGTCCTGGACTACGAGCACCAGACCCTAAAGAAAGAGCAGAACGGCCAG

[0255] CCCGCGCCCGCTGCCGGCCGTTTCCTGGACTTCGAATGGCGCGAAGGCTCCGGCCTGTGGGGGCGTGTCGAATACACCGCCCGCGCCGCG

[0256] AAGCTGATCGAGGACGGCGAGTACCTCTACTTCAGCCCTGTCTTCAGCTACGCCCCGGACGGCACGGTCCTCTCCATCCTCATGGGCGCA

[0257] ATGACAAATGACCCCGCCATCGACGGTCTGGAGCCCCTCGCACGCCGAGCGGCCGCGACCTTTGGCCTCTACCACCCCGACGAGGAAACC

[0258] CCTGTGGATGAACTCCTGAAAGCCATCATCGCGGCCCTGTCGCTGAAAGAAGGCACGACCGAGGCAGAGGCCATTGCGGCCCTGACCGCC

[0259] CTGAAGCCGGCCCTGGACGCCCAGGCCACCAACCTGGCCAAGCTGCGCGAAACCCTCGGCCTGGCCAAGGACGCGGACGTCGAGCAGATC

[0260] GCCGCCGCCACCGCCCAACTGAAGGTCGCCGCTCCCGGTAACCCTGACCCGGCGAAGTATGTGCCCGTTGAGGCCGTCACCCAGCTCCAA

[0261] GGCCAGGTCGCGGCGCTGACCTCTCGCCTCAACGGCGGCGAGCTGGACGGCCTGATCAACAGCGCTATCCAGGAGGGCCGACTCATTCCG

[0262] TCCATGGAGCCCTGGGCGCGTGAATACGGCGCCAAGGACTTGGCCGGTCTGAAGAGCTACCTGGGCCAGGCCAAGCCCATCGCCGCCCTG

[0263] ACCCAACAGCAGAGCGCCGGGCGTACCTCGGTGCCTACCTCGGTTGACCAGCTGGACGAGGCCGCCCTCGCGGTTTGCTCGGCTATGCAG

[0264] ATCAAACCGGAGGATTACCTCAAGACCCTGAAAGGCCAGTAAGGAGGCGCTATGACCGCCCTGACCACCGACCGCAACACCCCGCTCCAG

[0265] GACGCCGAGGTCATCGGCGTGCCGGTAGCGGCCAACGTCCAGGTCTTCGCCGGCGCCATCGTCGTGGCGAACGCTACCGGGTTCGCCGTA

[0266] GGCGGCAGCACCGCCACCGGCCTGACCTACCTGGGCCGCGCTGAGGAATACGTGGACAACCGCAATGGCGCGGACGGCGCCAAGGTCGTC

[0267] CGTGTGCGCCGCCTGAACGCCTTCAAGTGGGCGAACGACGGCAGCGTCACCCAGGCGCACCTGATGAAACCCGCCTACATCGTGGACGAC

[0268] CAGACCGTCGCCGCCACGGACGGCACCGAAACCCGCTCCCCGGCCGGCCGCATCATCGGCGTCGAACCGGACGGCGTGTGGGTGGAATAA

[0269] CAGGCTCACCAACGGAGAACCGGACACATGCTGATCAACAAGCAGAGTCTCAACGCGGCATTCGTCGCGATCAAAACCATCTTCAACAAC

[0270] GCCTTCGGGGCGGCCCCCACCACCTGGCAGAAGATCGCCATGGAAGTGCCGAGTAACACCGGCAGCAACGATTACAAATGGCTGAGCGCC

[0271] TTTCCGAAGATGCGCCGCTGGATCGGCGCGAAGGTGGTCAAAAACCTGAAAGCCTACAAGTACGTTGTCGAGAACGAGGACTTCGAGGCC

[0272] ACCGTCGAGGTGGACCGCAACGACATCGAGGACGACCAGCTCGGCATCTACTCGCCCCAGGCGCAGATGGCCGGTTTCTCGGCGGCGCAG

[0273] CTCCCGGACGAACTGGTCTACGAAGCGGTCAACGGTGCCTTCACGAAGCTCTGTTTCGACGGCCAGTATTTCATCGACACGGACCACCCT

[0274] GTCGGTGATGCCTCGGTGAGCAACAAGGGCACCGCTCCGCTTTCCAACGCCAACCAGGCGGCGGCGAAGGCAGGTTATGGCGCTGCTCGC

[0275] ACTGCGATGAAGAAGTTAAAGGACGAGGAAGGACGTTCCCTCAACGTCTCCCCCAACGTGCTCCTGGTCGGCCCGGCGCTGGAAGACGTG

[0276] GCGAAGATGCTACTCACCAACCCGAAGCTCGCGGACAACACCTCGAACCCCTACGTCGGCACTGCTGAGCTGGTAGTGGACGGGCGGATC

[0277] GAGTCCGACACCGCCTGGTTCCTGCTGGACACCACCAAGCCGGTAAAACCGTTCATCTACCAGCCCAGGAAGAAGCCGGTATTCGTTTCC

[0278] CAGGTCAACCTGGATTCGGATGACGTCTTCAACCTGCGCAAGCTGAAGTTCGGCGCCGAAGCCCGCGGTGCTGCCGGTTACGGCTTCTGG

[0279] CAGCTGGCCTACGGCTCCACCGGCACTGGCGCATAAGGGGGCAGCTCATGGCACGCAAAGCCGCAACTACCGCCAAGCCGACCGCCAGGC

[0280] AGGCCCGCCAGGCGGCGGGCGATGAAGCCCAGCAGAGCGTCGAGGGCATCTTCGTGCGCAGCTATCCGCCGACCTTCCGGCGTGCTGGCT

[0281] TCGCGTTCACCAGCGAAGGAATCGGAATCGCCCTGTCCGCCCTGACTGAGGCGCAGCTCAAGGCCATCAAGGAAGAGCCCCAGCTGCGCG

[0282] TCGAGTCCTGCGAGTTCTTGCCGGACGACGTCGGCGGCGAGGGTGAGCCGTCCGCGTCGGACGACGCCCCCCAGGAATAACCCACCCCAG

[0283] CAGAGGGACCGCCCCCGGCCACGGATGGCCACCTATTCATCAGGAGTTCGACATGAGCGACCACACCCTGGCCATCAGTCAACTGACCAT

[0284] CGCCGCGCAGAACGCCGAGCACAACGCTCCGATCATCGAAGCCCAGGGCGACCTCGCCCAGGCCGAGCTGGATCGCCGGGTCGCTGCCGA

[0285] GTGCCATAGCGCCATCGACGTCCTGGAGCACCAGGAGCCGCAACAGTGAGCTATTGCACCCAGGCCGACCTGGTCGAGCAGTACGGCGAG

[0286] GCGTCCATCCGCCAGTTGAGCGACCGCGTCAATAAACCCGCCACGACCATCGATCCGGCGGTCGTGGCCCAGGCTATCGCCGATGCGGAC

[0287] GCGGAGATCGATCTCCACCTGCACGCCCGCTACCAGCTGCCGCTGGCCCAGGTGCCTGTGGTGCTGAAGCGCGTAGCGTGCGTGCTGGCA

[0288] TTTGCCAACCTGCACACCCAGGTCAAGGACGACCACCCCGCGATCCTGGATGCCGAGCGCAAGCGGAAGCTGCTGGGCGGTATTTCCTCC

[0289] GGGAAGCTCAGCCTGGCCCTGACCAGCTCCGGCACCCCGGCGCCCATTGCTAACACCGTGCAAATCAGCTCGCAACGCAACGATTTCGGG

[0290] GGCACCTGGTGAGCACTGCCGAACCGTTCGATTACCTGTTCCTGGAGACGCTCCTGGTCGAGCGCATCCGCGCCGAGGTGCCTGGCCTCC

[0291] AGGATGTTTCGGGCATCCCCGATCTGGCCACCCTCGACGAACAGCCCCAGGGCTCGCCCTGCGTCTATGTCATCTACCTGGGCGACGGGA

[0292] TCGGCACCGGGGCGTCGCACCAGGGCGGTAGCCGGGCGATTCAGACGGTGACCCAGCACTGGGCGGCCGTGCTGGCGTTGTACTACGCCG

[0293] ACGCCCAGGGCGACGGACAGGGCGCCCGGCGCGAAGCCGGCCCGCTGCTCGGCCGGCTGCTCAAGGCACTGACCGGCTGGGTTCCTGACC

[0294] AGGGCGTCACCCCGCTGGCCCGCAGCCCGCAGGCTTCGCCAGTCAGCTACAGCAACGGGTTCTTCTATTACCCGCTGGTATTCACCGCCA

[0295] ACTTTGTCTTCCCGAGGCTCAAGTCATGGAAACCGTAAAAGTCACGATCACTGCTGAGAACCCCAACCACACCCACGCGGGCAAGCCGGT

[0296] GGCCAAGGGCGACGAAATCGAAGTCACCCGCGCTGCGGCCCAACTGCTGCTGCGCAAGAAGCTGATCGAAAAGATTCCGAAATCAGCGCA

[0297] AGGCTCGGCCGTTGCCGACAGCGACAAATAAGCCACCGAACTAGGAGGGTGCCGATATGGCCCAAGTCGAAACGTACTACTACGGCCAGG

[0298] GTAAGCTGAAGCTGGCCATCCATACCGCTACCGGGCTGACTCCGTGGCGCTGGCTGGGCGACGTATCCGCCCTCAGCGGTGCGATGGAAG

[0299] AAGAGAGGATGAGTCACCGCGAATCGTACTCAGGCTTGAAGGCAAAGGTGCGCGAGTGGGGCATTTCCCCCTCGTTGAACATGACCGCCA

[0300] CCCTGCACAGCGTGAGCGTGGAAAACGTCGCCCTGTTTTCCCAGGGCACCGCCAGCACCATCGCAGCCGGTACTGTGACCGGCGAGTTGC

[0301] TGCCGGCCGGCCTGGAGGCAGGCGACCAGGTTGCATTGGCGAACCCTGGCGTCAGCGACCTGGTGATCACTGACAGCGCGGGTACGCCCG

[0302] TCACGCTGACCGAGGATCACTACCTGCTGGAGCCTGCGTTTGGGGCGCTGGAAATCCTCAGTCTACCGACGTCGCCGGCTCCGACTCAGC

[0303] CGTTCATTGCCGCCTATGAGTACATGGCCCGCAAGCAGGTTGCGCTGCTGTCGGCGCCTAAGCGCTCCGATGTGGCCCTACGTTACGAAG

[0304] GCATCAACCTGGCTGAGGACGGCGCGCCGGTCATCATGGAGCTTTACAGGCTCTCCCCCGGCCTGCTCCAGGAACTGTCGATGATCACCG

[0305] ACGGCAACGACGTGGCGGGTATGCCTGTGAGCTTTTCCGCGCTCCGGGACACCTCCAGGCCGGCCAATGGTCCGTTGGGACAGTTCGGCC

[0306] GCATTATCCAGGTGGGCTAAGCCATGGCGAAGCCGAAGGCGAAAACGGCAGCTCCCGATGGCAGCCTGGAGGTTCTGTTTCCCGACCGTC

[0307] AACTGACGGTCGGGGGCGTCGAGGTGACCGTCCGCGAGCTGAGTTTCAGCGAGCAACTGCGCCACAACCATCTACTCGCGCCGCTTGGCG

[0308] ACAGCCTGGCCGCTATCCCCCCGCAGAAGATGGACAGCCCGGAGTCGATCAACGTCATCTTCGACGCCCTGGCCTTGCACGCTGACGCGC

[0309] TGCGCGAGCTGATCGCGATCAGCTGCGGCCAGTCTGTGGACTGGGTGGACACTTTGCCGCCAGACGAAGGTGAAGCCCTGGTGCTGACCT

[0310] GGTGGGAGGTGAACAGCGGTTTTTTCGTCCGACGGCTCTGGCGTCCGCGCCTCCTGGAAATGGCACTGGCCGCGCAGCGCTCTGGAGCCG

[0311] AGTCTTCGCCGACCTCATTCGCGCCGGCCACCACCGCGACGCCCTCAACAACTACACCCGGCGACAGCTGATTCTCTTTTGGGAAGAGGC

[0312] GCAAGCGGCTGAGCGCCGCGAGCAGGCCCGAGATATTAACGCGGTGATGTACGGAATGGTGGGAGGCACGGACGCCACCGCCTTGCTCCG

[0313] ACAGTTGGAATCCTGAAATGGCGAACAATCAGCAACTGACACTGGCCCTCCGCATCCGCGCCGATCTGAACGACGCCCAGGACGCGCTGC

[0314] GTCGTCTGAACGGTAATGTCGATGAACTGGGCCAGAGCACCAGGACAGCGGCGCGGGATGCTGATCAACTGAATGCCGCCACCCAGCGCA

[0315] TCGGTGGCGGCATGACGGCCCTTGCTGCCTCCATCAAGGCCGCTGCGCTGGGGCTGGGGGCGCTGTTCAGCATTCGGGAGATCATCCAGA

[0316] CCACCGACGCCTGGACCAACCTCCAGAACCGGCTGCGCCTGGTCACCAGCACCCAGGCTCAACTGGCGGAGGTGACCGCCGACGTATACC

[0317] GCATAGCTCAGCTGACCAGCTCCGTGCTGGGACTCTACCTCCGCCGTCTACCAGCGTTTCGCGCAGAACGCCGACTGGAACATCAGCC

[0318] AGCGGCAAGTCGCATCGCTGACTGAGACGGTGGCCAAGGCCGTCGCAATCTCCGGTGCCAGTGCCGCGAGTGCAGAGGCATCGCTTAATC

[0319] AGTTCGGGCAGGCGCTCGGCTCCGGTGTTTTTCGGGGTGACGAGTTCAACTCAGTTGCCGAGCAAACCCCAGCCCTCCATGAAGGCTATTG

[0320] CCGACGGCCTGAGTGTCACAACTGCCGAGCTTCGCAAGATGGCGGCCGAAGGCCAGTTGACGGCTGATGTCCTGGTGGACGCCTTGAGCA

[0321] ACGCAGCAGCCGGCGTGAACGATCAATTCGCCACCCGCATCAAGACCGTTTCGATGGCAGTCCAGGAGCTGGAGAATGCGTTCACTCGCC

[0322] TGGTAGGTGAGTTCACCAATGGCCGAGGTGCGGGCGAAGCGCTGGCCGGTGCCATTTCCGGGGTCGCCAATGTCATGGACGGCCTGAGCG

[0323] ATAACGCCGAGTTGCTGGGCACCGCCCTGGATGCCGTGATGGTGACGGCAGCCGGCCGAGCGGTGGCGGCCATCACTGGGCTGACTACTA

[0324] CCTGGCTCAGCAACGCGGCGGCCAGTCGCGCGCCAGCCATCGCCGCTCCAGAAAGCCACGCCGACGAAGGCGCCGCCGTGGCGGCAC

[0325] AGCGAGCCGCTGCCCAGGAGCTGCAACGGGCCAAGGCCGCTGTCGCCTCGGCAGAGGCCGAGGTAGCGGCCAGCCGTTCCCGCCAAGCCG

[0326] CAAGCCTCCAGAACCTTCGGGACGTCCAGGCCGCCCTGGTCGCGGAGCGCACGCTGGAGCAGGCCAGGCTTCAGGCGCAGATCACCGATA

[0327] TTGGTCGGCAGCAGTCCCTCGCTCGCCTGGCTGAGCTGCGCCTGTCCGAAGCGGCCATTATCAGGCAGGTCCAGGCCGCAGAGGCGGCGC

[0328] TCGCATCCACGACGCTGGCGTCTTCGGCCGCCGTCACCGCCGCCTATCAGCGTAGGACCGCCGCAGTCGCGGCCGCCGCTTCGGCACAAC

[0329] AGGCCCTGACGGTCGCCACGAACAACGCGAATATCGCCTCTGCGGCCGCCGCTGCGGCATCCAGCTTCCTGGCTCGCGGCATGGGAACAG

[0330] TGACGGCGGTCGGTGGCCGGCTGCTGGGGTTCCTGGGCGGTCCCATCGGCGTTATCTCGATGATTGCCATTGCGGCCACGGCGTTCCTGG

[0331] ACTTCGGTAGTGACGCCGAGTCCGGCATGGACCGTGCCGCCAATGCAACCGAGTCGGCTAGCGTCCGCATCCGCAACGCCTCGCGGAACA

[0332] TCATCCAGGCGCTGAACCTGGGTGACCTGAAGACGGCCAACTACGACCAGCTCGGCAAGAGCATCGAGCAGATCAAGCAGCAACTGGCCG

[0333] AGGCCGAGCAGATACAGCAGCGTGCCGAGGCCCTCCAGGACACGGACGTTCCTACGGTGCCGGGCATGGACCTCCCGAGCCTGGATGAAG

[0334] CCAACGAACGGGTACAGGCGCTGACCGGAGCCCTGCGCAGGCTTGAGGCAGAGCGAGCCGGCGACCGCTTCAAGAACGTGCGCGAGGGCA

[0335] AGCAGTACCTGGAGAACCTGGAGCGCCAGAACGAACGCCTCCAGAACCTCACCGCGACAGAAGAAGCCCTGAACTACCTGCGCAAGGAAG

[0336] GTATCGATGCCACGTCGGAGCTGGGTAGGAAGATCCTGGATCAGGCCGCCGCCAACCAGAAGCTGGACGCCACCAACAAGGCCGAAGCCG

[0337] AGTCCAAGCGCCAGTCTGAAGCGGCGGCGCGCAAGAGCGCTCAGACGTCGGACCAACTGCGCAAGAGCCAGGAGGGCTACGTCACCCAGC

[0338] TGGAACGCCAGGCGGCCCTGCTGGGTATGAATAGCGCCGAGGTTCGCGCCTACGAGCTGGCCGAGAAAGGACTTACCGGAGCGCTAAGGG

[0339] CCAGGGCAGAAGCTGCCCTTGCTGCTATCGATGCCGACGAGAAGAGGCGCCAAGCCGATGCCAATGCAAGCGCCAATGCCGACCTTCAGG

[0340] CCGAATATCTGCGGGCGACCGGGCGCGCCGTTGACGCGGGACTGCTGGAGATTAGGACGAAATTTGACGCGATGCGTCGGGACTTCGAAA

[0341] AGGCGGGCAACGACGCCGGCCTGGCCTGGATCGACAAGCTAATCCCCGTCGCCGAGGCCAAGGTCCGTCTGGATGACGTCAAGCAGAAGA

[0342] TGGACGACTTGCTGGCCGAGCAACAGCGAGCTGAGTCATCCGTCAACGTCCAGCAGGATGCCGGCGTTATCAACGAAATGGACGCTCGGC

[0343] AGCGCATCCTGGACATTCACCGAGCGACCTACGAGAAGCTCCAGCAGATTCGGCCGATCCTGGAACAGATGGCCCGCCAGCCTGGCGAGG

[0344] TCGGCCGAGCTGCTGCCGAATCGCTCGCCCAGCTGGAAGCTGAAGCGGAGCGACTCCAGCAGACCACGACGCTGCTCGAAACCACCTTGC

[0345] GGGACGGGCTCACTACCGGCTTTACCGACGCCATCAAGGGGCTGGCTTCCGGGACCATGGACTTGCGTGACGCCATTACGTCGCTGGGTG

[0346] AGGCTGTGCTCAATGCCTTGGTGAACATGGCGGCGCAGAACCTAGCGCAATCGCTGTCCAGCGGCATCATGGGGCTATTTGGTGGCGGTC

[0347] AGCAGGACACCAGCATGACGACCGGCGCGGCGGCTGTTACCGCATCGGCCGGTGCCCTATCGACTGCGGGCGCCTCGCTGCTGACTGGGG

[0348] CCGCTGCTATCCAAGCGGCCGCCGCATCCCTGGCCGCTGCTAACGGCGTCCAGGGCCTGGGCGCAGCCGGTGGCGCGGCGGGTGCGGCCG

[0349] GAGCAGCGGCGGGTGGCGGCAGTTGGTGGTCCTCCATCGCTGGAGCGTTCGGTTTTGCCAGCGGCGGCCATGTACGCGGCCCCGGTACGT

[0350] CCACCAGTGACAGCATTCCAGCTCGCCTGTCGGACTGGGAGTACGTCACCCGTGCCGCCGTCGTGAAGCAACCTGGCGCTCTGGCATTTC

[0351] TCGAACAGTTCAACCGTTACGGCATGGCCGCACTGGCCGGCTGGGCGAATCCTGTTCGTCACGCAACGGGCGGACGTGCCGGCATTCCCG

[0352] CCCCCAACCTGCCGGCCCCCGTCCGTGTCGGCGCGAACCTGCCCGAACCATCCAAGAACTTCAGTACCTCGGTCGCCAACTCGATCTACC

[0353] TGCACGCCGTTCAGGACACGGACCAGATGGCGGCCGAAATGTGGGCCGGCAAGGGCGGCGACCATTACATCGTCTGGCTGAACAAGAACC

[0354] GCCAGGCCGTCAAGCAAATACTCGGAAACTAGGAATCCATGGCTACTGAAATCGGCACTGCAACGAACCATGCTGACCTGGTCGAGCGCC

[0355] TCGTCCAGTTCCTCACCGCGAACCCAGACCTGGTCGCGGCTGGGCAGGCCTACGAGAAGATTTTCGACAACACCATCCCCGCGTCCGGCA

[0356] CGGCCATCGCCGTGCGCCAGGTGACACTGCGCGCCCCAGGCCTGGGCGGCACCGATGCCATCTACATGGGGATTCAGAGCTACGGCGATA

[0357] CCGCCCTGGACTACTACAACCTTCGCCTGATGGGCGGCACGGCGTTCAATCCTGGAGCAATCCCGCCTGGCGGCGACTACTGGACCGCGT

[0358] TTGCTAACTACAGCCCGCGGGTTCAGGCGCTGCTGTGGAATCAGCCCATGCCGTACTGGTTCTTCGCCAACGGCCGCAGGTTCTGGCTGG

[0359] TCGCAAAAGTCTCGACGATCTACGAGTCGGCCGGCGCCGGCTTCATCCTGCCACCCTGTCCGCCGTCGCAGTATCCGTACCCGCTGGCGG

[0360] TGGTGGGTTCCTACCGTGGCGACGTCGCCACCCGCTGGTCCGACGTGAGCGACCGGCACCGAGGCATCAGTAGCCCGCTTGAGCGCAGTT

[0361] GCTATGTCCGCGACCCGGCGGGCCGTTGGCTGGGCTTCACCGTCGCATCGAACAGCAACAACGAGTCCGACTACAACAATCGGACCCTGC

[0362] TGCCGCTGGGCTGCGCCCGCTATGCGGGTAGTAGCGACACTGTGATCAACCAGCTCCGAGACTCTTTCAGCAAGTTCCCCCTGAAAGCAC

[0363] TGTCGTTCGTCACCCGCGAAACCGAGGGACGCCGCTACCTGGGCGATTTCGACGGCGCCTTCTACGTGCCGACGCTCAACTCCGGCGCCG

[0364] AAGACGTGATCGTCGAGGACGGTGTGGACCACGTCGTTTTCCAAACCGCTTGGCGTAGCGGTAACCCTTGGCTCTACGCGATCAGGAAGG

[0365] ACTGACATGGCCTATTTCACAGGAACAGCGAACAACCCGGCCGACTTGCTCGCCAAGCTGCGCGTTCACGCCGAGTCGCTCGGCTGGGTC

[0366] ACCGACCGCGCCTCGGCATCGGAATGGCTTTGTCACAACGCCGACGGCTACTGGTCGTTCAACGCCGGTTCCAATCAATGGCAGCTCGCC

[0367] GGCAATACGGGGTTCGACAACGCTTTGGCGTGGAACGCGCAGCCAGGCAATTCCGTACAGAACAATCCTTATTCGTCAAAAGGGCCGACC

[0368] ATCGCGCAGCTCAGCGGCGGGCCGTTCACTCGTTACCACCTGTTTGCTACCGCTGCGTATCTGCACTTACACGTCGAAATCGCGGCAGGT

[0369] CAGTTCCGGCCGGTGATGATTGGCTCGCTCAACAAACGGGGCGTCGGCTATACGGGCGGTCAGTATGTCTGCGGCTCGTTCATCTATACC

[0370] CCCGGTCAGGCACTGACAAACAACTGGTCGTCGCATCCGTTCGATGGCTACCACATTCAATACAGCAACAGCAGCTGCATGCTGCGGCTG

[0371] GACGGCCTCGACGGCGGCTCGTCGCCGGAGTGGTTGCCGTTCGACTACACAACGAACGTCCCCCGGCGCGTCGTCGGCCCCGGTCGCGGA

[0372] AACTACAGCAGTCAGTACCATCCCGACGTGGGGCTGATCGACGCCAGCGCAAACGAGCTGAACAGCTCGACCACCCCTGTGCCCTGCGCC

[0373] ATCTATGCGTTCGGCGCTCAGCAGCGCTCGCGGTACGTGGGCGAGGTGCCGGACTTTGGCATATGCAACATGGCCTTCCTCGCGCCTGGT

[0374] GATCCGCTTGTCGTCGGTAGCGACACTTGGCGCGTCTATCCGTTGCTCCAGCGCGGAACCGCTACCGACTTCGGCGCCACAAGCGCCTGG

[0375] GTCGGCTATTGCTTCCGGGTGGTCGAGTGATGGCGACCTTTCCGGGGTTCCAAGTGCCAAAGCCGCTCGTGGGGATCGTTGCCGGCATCA

[0376] CGCCGAATATCGACGCCCTGGAGCTGAACCAGGACATCAGCCTGGCAGCGGTCGCGGCCTCGACCTGGGCCGGCGCCTACGCGGCGCATC

[0377] AGCCGGTAGAGGTGATCCATTCGACGTACCAGGCTGTCCACCAAAGCGCTCTGGAAGAGAACTACTACAACCGCCTGTGGCTGATCCCTA

[0378] CGATCATGGAGCTGGGCAACGTCGTCAGCACCCAGGTACGACCGGCATCAGTCTGGAACGCTTATTTCAGTCCGCGCACGCTGACCGCCA

[0379] TCGAGCGGGAAGATGCAGACGGCATCACGCTATCCGGCCAGGCGTCGCCGCCGCTGGGTTTTGCCGCCCTGGAGGAACGCACCTGGACGG

[0380] TCAGCATTGGCACGGACGGCCCGCCCGTCGTCAATGCGCGGATCGTCTGGAGGCTCCAGGGCGAGCCGGAGCTGGTCCTGGTCATCACCG

[0381] GCAATCGGATCATCGCCTGGACGTTCGCACCGGACTGGGGCGACAGCATCGTCGAGCGCCTGAGCGCCTCGACAAATATCCTGCAAAGCG

[0382] AATCAGCCGTGACCCAGCGCCGGGCTATGCGCCTGGCGCCGCGCCGAGAATTCGAAGCGAGCATGTACGCGGTTGACCGCGAGCGGCAGC

[0383] TGCTGGACATGACGCTGTTCGGCTGGGGCGCACGCATTTGGGCGCTGCCGATTTGGCCTGATATCCAACTGCTCCAGGAACCGCTGGCGG

[0384] CCGGCTCGCTGAACATTCCGTGCGGCACGGCCGGCCTCGACTTCCGCGACGGCGGTCTGGCGATGCTGCGCGGCGAGGACGCTTTTACTT

[0385] ACGAGGTCGTCGAGGTCAAGACGGTGACCGCCAGCGGCCTGGACCTGGTCCGCCCCGTCCAGGCCACCTGGGTAACTGGCTCGCGACTGT

[0386] ACCCGGTACGCACCGCGCAGCTGACAGAACAGCCCACGCTGACCCGGCTGACCGATACCGCGCAGTCTGCGCGGGTGTCGTTCCTGATAA

[0387] TGGAGCCCAGCGCCTGGCCCGAGGTAATGCCGGCGACGACCTACAGGGGGCGGCCAGTGCTGGAGCAGCGCCCGGACGAAAGCGAAGACC

[0388] TGACGTCGAGCTATCAACGCCTGCTATCGACCCTCGACAACGGCAGCGCCATTCCCCGCGTGACCGACGTTGCCGGCATTGCGCTGCCGG

[0389] TCATCGGCCATCGCTGGATCGGCATGGGCCGAGCCGAGCGGTCGACGTTCCGTAGCCTTGTCTATGCGCTGCGCGGCCAGCAGAAGCCGC

[0390] TATGGGTGCCGACCCACGCCGACGACCTGACTCTGGTCGCCACCGTCTCGCAGCTGTCCACCGCCTTGGACGTGCGCAATATCGGCTATG

[0391] CCCGTTTCGCCAATGGCCGGCCGGGCCGGCGCGACATCCGCATCGAGCTGTACGACGGCACGGTCTATCACCGCCGCATCCTCACCAGCA

[0392] CCGAGCTGGACGCCGACACCGAGCGCTTGGCCATCGACGCCGCCCTGGGCCGACTGGTCGAGCCTGGCGACGTGGCGCGCATCTGTTTCA

[0393] TGGCGCTCTGTAGCGCCGCCACCGACGTGGTCGAGATTGAGCACGTCACCGATAGCGAGGGCGTCGCAACTGCCGCCCTGACGTTCAAAG

[0394] GGGTTCGAGACGATGAGTTTTAACAGCCGCGAACGCTCGCTCGCGGATGGGCAGCCGGTGCGGCTGTACCAATTCAGCCGTGGCGCGATC

[0395] CGCTGGAGCTACAACAGCAGCGACCGGGACATCACCTACCAGAACCAGATTTTCCGCACGGTGCCGGGCGGCATCACCGACAACGGGATT

[0396] ATCTGTTCCGGCGATCCGCAGTCCGACCAGTTCGTCATCACCGCGCCGGCCGACCTCGATGTCGCGCTGCTGTACAAAATCAGATCGCCG

[0397] AGCGGTTCCGTCGACCTGGTCGTCTACGACATGCACTACGGCGACGCCGAGGCAGCGGTTTCCTGGGTGGGCCAGATTGGCGATGTGGAC

[0398] TGGCCGACCGTGGATAGCTGCCGCATAACGTGCGTGTCCGAAGACGAGCTGATGGACCAGCCCGGCTTGATCGACACCTACTGCCGCACC

[0399] TGCACGGCAGTCGTTGGCGACCATCGCTGCAAGGTCAACCTCGTTCCGTATCGCGTGACGCTGACGCCGCAGAGCATCAGCGGCTGGGTG

[0400] ATCTCCAGCGGCGTGGTCGCCGGTTATGCCGATGGCTGGTTTACCGGGGGCTACGTCGAGTGGCAGGTGGACGGCGACAACTACGATAGC

[0401] CGCTACATCGAGCGGCACGCCGGACCCGATCTTTACATCCTGGGCGGCACCGAGGGCATTCCGGCAGGTGGCCAACTGCGGGTTTACCCA

[0402] GGTTGCGACGGGCTCGCGCAGACCTGCAACGACAAGTTCAGCAACCTCCCCAACTTCAGGGGGTTTAACGCGATGCAAGGCAAGTCGCCG

[0403] TTCGATGGCGATCAGGTCTGGTGAGGTAGGCCATGGACCCGATCACAATCAATCTCGTCATCTTGGCGGCGTCGTTCATCCTATCCAAGG

[0404] TCTTGGCGCCGAAGCCGCAGAAGCCCAAGCCGACCGCCTTTGAAGACATCGATTTCCCGCGCTGTGACGAGGGCGACGAACAGGTCGCCG

[0405] TCTTCGGTCAGTGCTGGTCGAAGAGCTGGATGGTGCTGACCGTGGGCAACTACAGAACAAAGGCGATCAAGACCAAAGGGAGCAAGAAAT

[0406] GATCGTTACGGCTCAGCACCTGCATACCGTGCCGACCTGGACCACTCGGCAGGGCTACTGCCACCGGCAGGCGCGAGACTTCTTCAAGCG

[0407] CCATGGCCTGGATTGGATGGCTTTCCTACGGGACGGCATCGAGGCCGACGTGCTTGTCGCGACCGGCGACGCGCTCGCGCTCAAATTGGT

[0408] TGAACATGCACGCCTGGAGGTGGCCGATGGGCGCTAAACCGAAGGCGCAAACGGTCGGGTTCGAATACTTTTTTGACATCCATTTCGCCC

[0409] TGGGTAAGAAGATCGACGAGGTCTGTGCAATCCGGGCGAGCGGAAAGACCGCATGGAAGGGCTCGATCACAAGTAACGGCCAGGTTCGAA

[0410] TCAATGCGCCGGACCTGTTTGGTGGGAAAAAGGGCGAAGGCGGCCTCGACGGAACGCTTGACGTGCTGTTTGGCGAAGAGGACCAGGGCG

[0411] TCCTGCCTCGCTTGGCGGCCATGCTCGGCGGCCTGGTGCCGGCTTTCCGGGGCGTTACCACGTGCTTCTATTCCGGCCTGGTCACCGCCA

[0412] TGAACCCCTACCCGAAGACCTGGGAGATTCTGCGCCGAGGTGGCAACCGCCTGTGGGACGGCAACCCCTGGTATCCCGAAAAGCAATTTA

[0413] TCTGGCTCGCGGACGGTCAGATCAAAGCGATGAACCCGGCGCACATCCTTTATCTCGTCTACACCGGCCGGGACTTCCGGGGGCTGGCCC

[0414] GCACGCGGATGGACGAGGCGAGCTGGCGGGTCGCTGCCGACAAGCTGTATGCCGAGGGTTTCGGGCTGTGCTTTGAATGGACCAGGTCCG

[0415] ACACGTTCGCCAACTTCTGCGAGACGGTGAAATCGCATATCGGAGCCGAGGTCTACCCGAACCGCCAGACTGGGCAGATCAGCATCCGGC

[0416] TCCTGCGTGACGACTACAACGTTGCTGACCTGCCGCTGTTCGACGAAGACAGCGGCCTCCTGGAGATCACCCAGGAGAAGACCGGCTCGA

[0417] CCTCGCTCGCGCCGAGCCAGCTTATCGTCAAGTACATCGACCAGACCGACGGTGCACAGCGCCAAGTCATCGTCAACAACAACGCGGTCG

[0418] CCGCGTCGCAGGGGCGGCGGTCGTCCGAGGAAGTCGAGTTCCTGGGCGTGCCGACTGGCGAGCTGGCCGGGCGAGTCGGTGAGCGGGAAA

[0419] TGCGTCTGAAGACAACCGGCCTGAAGCGCTATAAAGGCGTATTCGACCGCCGCGCCCGTAGCCTGAACCCAGGCCAGCCGTTTCGCATCC

[0420] GTTCGACCCGGCGCGGCATCCCCGAAACCGTCGTCAGGGTCGGCCGGATCGAGGACAACTTCCTCGGCGACGGCAAGATCACCCTGACCG

[0421] TCGTTCAGGATCAGTTCAATCTGCCGGCGACAACCGGCGTGGCGCCGCCACCACCGGGCTGGATTCCGCCCGACCGGACGCCTCGGGCGA

[0422] TCACTGTGCGCCGCCTGATCGAGGCACCCTATCGCGAACTGGCCGGCGTGATCGATCCGGCGAATCTCCAGCTCCTGGACGTGAGCGCGT

[0423] CCTACTTGGCCGCGCTGGCCGAGGCGCCGACCAGCCTGTCGCAGAGCTACACCTTGACCGACCGCGTCGGCAGTTCGGGCGCGTTCGTTG

[0424] ATCGCGGAACCGGGGATTGGTGCCCGACTGGACTACTCGCCGCAGAGCTGCCGCTGGCGGCCGGTCCGAACGTCGTCACGTTGACGAACG

[0425] CCAGCCGGCTGGAGGACGTAGCTGTCGGCCAGGCGGCCGTGGTGGATGACGAAATCGTCCGGGTCGATGCGGTCAACTACGCCAGCGGTA

[0426] CTGTCACCCTGGCGCGCGGCTGCGCCGATACCGTACCAGCCAAGCACCTGGCCGGGGCTCGGGTCTGGTTCTATGACACGTTCGAAGCGG

[0427] TGGACGAGACGGTCTACAGCCAGGGCGTGACGCTCCAGGCCCGGCTGCTGACCAACACCAGCGAGGGCCAACTGGCCCCGGCGCTGGCCG

[0428] CCACTGACAGCCTGACGCTGACCGGGCGCCAAGGTAAGCCGTACCCGCCCGGCCAGTTCCGCATTAACGGTAGCGCGTACCCGGCCAAGG

[0429] TCTACGGGGTGCTGTCGGTGAGCTGGGCGAAGCGCGACCGGATCGGCCAGGCCGACCAGTTGATCGATACCACTGTCGGCAACATCGGGC

[0430] CCGAAGATGGAGCGACGGTGACGCTCCAGGTCTACAGCGGCACGACGCTGAAGCGCACCTATGCCGGCCTCACATCCAGTAACTGGTCCT

[0431] ATCCGGTGGCCGAAGACGTCGCTGACGGCCCGCTCCAGGACGTGCGCCTGGTCCTGCGCAGCGTCCGCGACGGCATCCAATCCTGGCAGC

[0432] AACACGACATCACCATCGAACGACACGGCCTGGGCTTCCGCTTGGGCGAAGACCTTGGAGGCGTTTCCGCATGACTCTCTATATGGGGCC

[0433] GAATACCGGCCTGCTGATCAATGGCTTGCCGGGAGAAGGGCATTACAGCGATCTGATTCGGATGTGGCGCTGGGATGACTTCCTGCGCCA

[0434] ACCGGTCGTCAAGGGGCGCGTCGCCACACTGCCCACAACCGGCCAGGCCGAGGGGGACACGTACATTTTCACTGGCTCCGGCTCCAATCA

[0435] GAACCGCCTAGCGCGCTGGTGGGCAACGGGCGCCACCACGGCAATTTGGGAGTACATGCCGCCACGGCTGGGCTGGCGTGTCCAGGTCGC

[0436] AAACGAGACGACGCCGAGCGGCCAGGTCAAGACGTATGAGTATTCCGGCACGGCGTGGGTCGAGCTGGTGGGCGGTATGTCGGACGCGCC

[0437] GAGCGACGGAAAAGCGTATGCCCGCGAAAGCGGCGCCTGGACGGAGCTGGGATCGGCCGCGAAATCGGCGCTCAACGTTCTGCCGTTCAT

[0438] GAACCTGATGCCCGACATGGGCCGCTTCGCGGGAACCGCAGCCAACCCTCTGGCTACGATGTTCACAACGTCATGGACTCCAAGCTCGTT

[0439] TCTCAACGGCTGGAACGGCGCCACCGTTGCAGATGGGGGCAAGTTCGCCTTCGACAACAGCACGAACGGCGGGGCCGGGCCGGCGCTCAA

[0440] TGCGCGGGTGCAGGCGCTGCTTGCGGCAATGGGCCGGACGTGGACGTCGGTGTCACGATACGGGGTCGAGTTCTTCACCACCGTTCTGAC

[0441] GGCGGGATCGCAGACAACAACTGGCTCGGCCGGCGCGGATGGGGTTACGCGATATCTGTGCTGCTCCAACGGCAGCAAAACCGTTTTCAA

[0442] CGCGGGCGCATGGGCGACGGTGGTCATGTGGCTGCGTGTCGAAAGCGGTTCGGCTCATATCTCGTCGGCGCCTTATACCACCCATCGCCT

[0443] TTGGATCAATGGCGCTGTCGCTGCTCCTGGCGTGGTGCTGCCTGCGAACCAATGGGTGCATCTGCGATTCTCGATGCAGTCATACAACGG

[0444] CTATGACAACGCGTGTCCGTACATCTACGCATCCGCAGGGGCTCAGATCGCGTTCGCATGCCCGGCGTGGTTCGGTGGCCTCGTCGATCC

[0445] AGGCATCCACGTTGCCCCCATCCTCACCATCAACGGAGCAAGCGCATGATGAAGCGAGTTCTCTACAAAGGGGCGTTCTTCACTGAGTGG

[0446] GACGGCCCGCTGGCGGCCATCGCTGAAACTGCCGGAGTGAGCATGGACGATCTCGCGTTCCACCCGGATGACGCGCTGGCGGAGGTCCAG

[0447] GAACTGCGCCGGCAGGCATACCGGGACGAGGCAGACCCGCTGCGCCTGGAGGCCGAGTATGACGCCATCGCTACCGGCACAGCGCCGGAC

[0448] CTGTCGGCCTGGGTCGCAGCTGTCAAGGCGATCAAAGAGCGCTATCCGCTCCCCCAGTCCTAAGCGTTTTGATAATTGTGACCAACGTCT

[0449] CCTTTTTGCTACGGTCCCCCGCTGATGTGCGGAGTAGATAGGGATGTTGGTATGAACGAGATGCTTAGACGGAGGCTGCGGGCGGAGCTG

[0450] CTGGAGGTGGGGTTTCTCAACCAGTGCTGCCTTGATCTGATGGAAGCGATGGAGGCCGAGTTCAGCCTCACCGAGGACCAGCGCGAATGC

[0451] ATCGAGCAGCTCAGCCGATTCCTGCGGGAGGGCATCGGCAAGCTGACCGCTCTGTCTGAGCGGGTAGCCGATGGCGATATCGTCGTCCTG

[0452] TGCTGATCTTTTGAAATTCTTTTGCCGCTGGCGAAACGGTTAGGGCGCGTCATTTATTGCGCAAATCCGCGCCAAATTTCGCGCCGCGCT

[0453] ACACGGTGTCGCCGCTGCTGCGGCAACTGTTGCTGGCGGCGGTGGACATGCCCCTGGAATACGACACCGAAGGTCGCGACGGCGCGTTGG

[0454] CGACGCTGTTGCTCCATGAGCTGGCGGCAGCCGAGACGCTGCCGCTGCACGTGCCCTTGCCGCCCGAGCCGCGCTGGCTGGCGCTCTGCG

[0455] AGGCGTTCCTCGTCGAGCCCGACATCCGCGCCAGCGCCGAACGCTGGGCCGGGTTGCTGCATACCAGCCTGCGCAGTTTCAACCGCACCT

[0456] TCCGTCGCTACACCGGCCTCAGCTTCGGTGCCTGGAAGCAACGCGCCTGCGTGGTGCAGGCACTGGCGCGGCTGGCCGGCGGCGAAACGG

[0457] TCACCGCCATCGCCCTGGACTGCGGCTACCAGAGCCCGGCAGCCTTCTCGACACCATGTTCCGCCGGGTCCTCGGCCAGCCGCCCAGGCCT

[0458] TCCAGGCGGGCGCGCTCAGATCGCCAGGAGGTCCATGAAGCGGTTCACCGGCGTGGCTTCCAGCGATGCCTGGTGGCTGCACAGGTCGAA

[0459] GATCCTCTGGCAGCGCTGCGGCGGGAAG

[0460] The GC content is 64.6%, and there are no antibiotic resistance genes and virulence factors in the genome. From the map, it can be seen that phage J3 has a total of 57 protein-coding genes, including 25 hypothetical proteins, 4 unknown proteins, and 28 known functional proteins.

[0461] 4. Phylogenetic tree analysis of bacteriophage J3

[0462] The NCBI BLAST database was used for analysis, and the whole genome sequences of 15 phages with high homology were compared to perform a phylogenetic tree analysis of the large subunit of the phage terminase. The ML method (Maximum Likelihood method) in the MEGA11 software was used to construct a phylogenetic tree based on the large subunit of the phage terminase, and a phylogenetic analysis was performed on the whole genome sequence of J3. The results are shown in Figure 2. Figure 3 , indicating that phage J3 is closely related to Pseudomonas aeruginosa phage F_ET309sp / Pa1651 and vB_PaeS_PAO1_HW12, and is distantly related to other phages.

[0463] 5. Observation of morphological characteristics of bacteriophage J3

[0464] Phage morphology was observed using the phosphotungstic acid negative staining method. 1 mL of phage was centrifuged at 4°C, 200 μL of 2% glutaraldehyde was added to the supernatant, mixed and allowed to stand at 4°C for 30 min, the supernatant was centrifuged, and the phage was resuspended in 100 μL PBS. A small amount of concentrated phage sample was placed on a carbon-coated copper grid to absorb for 15 min, then stained with phosphotungstic acid (PTA, 2% w / v) for 3-5 min, and observed and photographed using a transmission electron microscope. Figure 4 As shown, phage J3 is a long-tailed phage with a polyhedral head with a diameter of about 63.27 nm and a tail length of about 190.12 nm.

[0465] VI. Lysis Kinetics of Bacteriophage J3

[0466] Pick a single plaque and inoculate it at OD 600 The enriched solution was centrifuged at 4000 rpm for 5 min and filtered through a 0.22 μm pore size sterile filter to obtain the phage J3 filtrate. The PAO1 bacterial solution was diluted to an OD of 0.3. 600 The ratio of PAO1 bacterial solution and J3 phage solution was 0.3, and the PAO1 bacterial solution and J3 phage solution were added to a 96-well plate in equal proportions. The PAO1 bacterial solution was used as a control, and the LB medium was used as a blank. Each well was repeated at least 3 times, and the kinetics was measured for 20 hours. Figure 5 After 20 h of culture, the OD of the PAO1 bacterial solution alone was 600 The OD of the bacterial solution containing J3 reached 2 or above. 600 is about 1.3, which indicates that phage J3 can effectively inhibit the growth of host bacteria.

[0467] VII. Determination of phage J3 titer

[0468] Pick a single plaque J3 and inoculate it at OD 600The enriched solution was centrifuged at 4000rpm for 5 minutes and filtered with a 0.22μm pore size sterile filter to obtain the phage J3 filtrate. The J3 filtrate was diluted with LB medium in multiples, and the logarithmic phase PAO1 bacterial solution was mixed with phage J3 of each dilution in equal proportions, and then mixed with solid culture medium and poured into LB double plates, placed at 37℃ for culture, and the titer was calculated as follows. The titer of J3 was calculated to be 1×10 12 PFU / mL.

[0469] Titer (PFU / mL) = number of plaques × dilution factor × 10

[0470] 8. Determination of the optimal MOI of bacteriophage J3

[0471] A single colony of PAO1 was inoculated into LB liquid medium and cultured at 37°C until the logarithmic phase. It was diluted 10 times in a gradient manner, and 100uL of the PAO1 dilution was plated for overnight culture. The number of colonies on the plate was calculated. PAO1 was cultured until OD 600 is 0.1, at which point the PAO1 concentration is 1×10 8 CFU / mL, J3 was diluted to the corresponding concentration according to the infection multiplicity MOI of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001, and the J3 dilution and PAO1 dilution were mixed in LB in equal proportions and cultured at 37°C for 4 hours. The culture solution was centrifuged and filtered to obtain the filtrate. After appropriate dilution, the phage titer was determined using the double-layer plate method. The highest titer ratio was used as the optimal phage infection multiplicity. Repeat 3 times. The results are as follows Figure 6 The titer of phage J3 is 1×10 12 PFU / mL, the number of bacteria in PAO1 is 1×10 8 PFU / mL, the optimal infection multiplicity MOI of phage J3 is 0.1, at which the phage titer is 7.11×10 10 PFU / mL.

[0472] IX. One-step growth curve of bacteriophage J3

[0473] A single colony PAO1 was picked and inoculated into LB, and cultured at 37°C until the logarithmic phase. According to the optimal multiplicity of infection MOI of 0.1, J3 and PAO1 were diluted and mixed in equal proportions and allowed to stand for 2 minutes to allow the phage to fully adsorb to the bacterial surface. The mixture was centrifuged at 4000rpm for 5 minutes, and the phage supernatant that failed to adsorb was removed. 1mL LB was added to wash and resuspended twice. 1mL of the resuspension was added to LB, and gently pipetted with a pipette to mix. The mixture was cultured at 37°C with shaking and timing. Samples were taken at different time points, and the phage titer was determined by the double-layer plate method after appropriate dilution. A one-step growth curve was drawn with titer as the vertical axis and culture time as the horizontal axis, and repeated 3 times. The results are shown in the figure. Figure 7 As shown, the incubation period of phage J3 was 60 min, the lytic period was 30 min, and then it entered the stationary period.

[0474] 10. Effect of temperature on the stability of bacteriophage J3

[0475] The phage solution with determined titer was added to a centrifuge tube and incubated at -80℃, -20℃, 4℃, 28℃, 37℃, 40℃, 50℃, 60℃, 70℃ and 80℃ for 1h. After appropriate gradient dilution, the phage titer was determined by the double-layer plate method. With temperature as the horizontal axis and phage titer as the vertical axis, GraphPad Prism9 was used to draw a curve of the effect of temperature on phage stability. Repeat 3 times. The results are as follows: Figure 8 The optimum temperature of J3 is -20℃~40℃. Low temperature has little effect on the phage titer, but the phage titer is significantly reduced when the temperature is higher than 50℃.

[0476] XI. Effect of pH on the stability of bacteriophage J3

[0477] Use 1 mol / L HCl and 0.1 mol / L NaOH to adjust the LB liquid culture medium to 14 different pH values, namely 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0. The phage with determined titer was mixed with LB of different pH values, incubated at 37°C for 1 hour, and the phage titer was determined by double-layer plate method after appropriate gradient dilution. GraphPad Prism9 was used to draw a curve of the effect of temperature on phage stability, with pH as the horizontal axis and phage titer as the vertical axis. Repeat 3 times. The results are as follows: Fig. 9 J3 can maintain good activity at pH 6-9 and has a wide tolerance range.

[0478] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A Pseudomonas aeruginosa phage, named phage J3, was deposited in Guangdong Microbiological Culture Collection Center on May 16, 2024, with the deposit number GDMCC No: 64627-B1, and the taxonomic name: Pseudomonas aeruginosa phage.

2. A drug or biological preparation for preventing or treating Pseudomonas aeruginosa infectious diseases, comprising the Pseudomonas aeruginosa phage according to claim 1.

3. The drug or biological preparation according to claim 2, characterized in that: The medicine or biological preparation uses bacteriophage J3 as the only active ingredient.

4. Use of the Pseudomonas aeruginosa phage according to claim 1 in the preparation of a medicine or biological preparation for preventing or treating Pseudomonas aeruginosa infectious diseases.

5. Use of the Pseudomonas aeruginosa phage according to claim 1 in preparing a phage disinfectant or inhibitor whose host bacteria is Pseudomonas aeruginosa.

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