CRISPR / Cas9-based preparation method, vaccine strain and application

Abstract

The invention provides a preparation method of a vaccinia virus gene modified vaccine strain based on CRISPR / Cas9, the vaccinia virus gene modified vaccine strain based on CRISPR / Cas9 and application of the vaccinia virus gene modified vaccine strain. According to the present invention, the C7L gene is inserted back while the F1L gene is deleted, the modified NTV modified strain obtains the expected result, the mouse virulence experiment results prove that the virulence of the NTV modified strain is close to the virulence of the NTV, and is significantly lower than the virulence of the vaccinia virus Tian Tan strain TTV, the humoral immune response level and the cellular immune response level induced in the mouse body through muscle immunization are higher than the virulence of the NTV, and the statistical significance is provided. The NTV derivative strain is expected to become a candidate strain of a substitute product of a current Cheng / monkey pox vaccine in China, and is subjected to pilot plant test and clinical test research and development.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for preparing a CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain, and its vaccine strain and application. Background Art

[0002] Vaccinia virus, smallpox virus, and monkeypox virus are all members of the Orthopoxvirus genus of the Poxviridae family. In the Orthopoxvirus genus, the gene homology of its members (viruses) is greater than 90%. The viruses that infect humans in the Orthopoxvirus genus mainly include smallpox, monkeypox, cowpox, and vaccinia viruses. Among them, vaccinia virus was once widely used as a smallpox vaccine for human vaccination and played a key role in the process of eradicating smallpox virus. Since the World Health Organization announced the eradication of smallpox in 1980, vaccinia virus has been widely used as a gene expression vector in tumor treatment and vaccine development for various infectious diseases, such as AIDS, tuberculosis, and new crown.

[0003] Monkeypox is a zoonosis caused by infection with the monkeypox virus. Over the past fifty years, the disease has mainly occurred in the tropical rainforests of central and western Africa. In early May this year (2022), the United Kingdom reported the first confirmed case of monkeypox without a recent history of travel to African countries. In just a few months, the number of monkeypox cases in Europe, the United States and even the world has surged. At present, the monkeypox epidemic has shown characteristics such as a wide range of spread, rapid spread, unclear source of the epidemic, and mainly human-induced transmission. It has become a new threat and challenge facing human public health. Governments of various countries are actively looking for response strategies to control the epidemic, and vaccination is the main measure for monkeypox.

[0004] Since monkeypox virus also belongs to the genus Orthopoxvirus, the vaccinia virus used to prevent smallpox should be able to cross-protect against monkeypox. Studies have shown that the cross-protection rate of the second-generation smallpox vaccine in the United States against monkeypox reached 85%. The cross-protection of the third-generation smallpox vaccine in the United States (replication-deficient vaccinia virus MVA-BN) against monkeypox has completed phase III clinical evaluation, but a larger-scale evaluation is still needed. Currently, the United States has approved the third-generation vaccine Jynneos (MVA-BN, replication-deficient vaccinia virus, subcutaneous injection, which can be used in immunocompromised people, such as AIDS patients) for the prevention of monkeypox. However, the United States only recommends this vaccine for people at risk of occupational exposure to orthopoxviruses (such as smallpox and monkeypox). For the first time vaccine recipients (no history of smallpox vaccination), two doses are subcutaneously injected, 4 weeks apart, and those with a history of smallpox vaccination only receive one dose. Countries such as the United Kingdom recommend this vaccine for post-exposure prevention of unprotected high-risk exposed persons.

[0005] However, the research on the replacement products of smallpox vaccine in China has been relatively slow. At present, the vaccine used by my country National Biotec Group for smallpox emergency reserve is still the first generation product, which is inoculated by skin scratches, has high side effects and is not suitable for people with low immune function such as AIDS patients. As early as around 2000, Professor Ruan Li of the Institute of Viral Diseases and others independently developed the non-replicating (or replication-deficient) vaccinia virus Tiantan strain (NTV) based on the vaccinia virus Tiantan strain of my country's smallpox vaccine. Its virulence is significantly lower than that of the vaccinia virus Tiantan strain (TTV), and the immune effect is reduced, but the immune effect can be improved by increasing the immune dose. In 2010, the Institute of Viral Diseases cooperated with enterprises to carry out pilot production and research and development of NTV as a replacement product of my country's smallpox vaccine, but it has not been approved for Phase I clinical trials so far.

[0006] In recent years of research, the inventors found that the expression of late proteins of the non-replicating vaccinia virus Tiantan strain (NTV) in human cells was significantly blocked, and there are literature reports that the late proteins of the foreign replication-deficient vaccinia virus MVA can be normally expressed after infecting human cells. The inventors speculated that the immunogenicity of NTV is likely to be inferior to that of MVA. Therefore, in the research of smallpox vaccine alternatives, the inventors performed genetic modification on the basis of the non-replicating vaccinia virus Tiantan strain NTV in the hope of improving its immunogenicity without changing its virulence.

[0007] At present, the main transformation direction of vaccinia virus as a vaccine vector in the world is focused on anti-apoptosis genes. The possible reason is that the anti-apoptosis gene of vaccinia virus is related to the virulence or immunogenicity of the virus. There are 6 anti-apoptosis genes of vaccinia virus found so far, namely B13R, C12L, F1L, N1L, E3L and M1L. Among them, C12L, N1L and M1L have been artificially removed in the process of constructing NTV. The E3L gene is related to the virulence and host range of the virus, which can ensure the translation of the viral mid-term protein mRNA. Its deletion will result in the inability to produce mature progeny viruses. After sequencing and identification by the inventors, it was found that the ORF of the B13R gene was broken and could not express functionally active proteins. Therefore, the inventors focused their transformation on the F1L gene. The F1 protein is expressed in the early stage of viral infection and is localized to the mitochondria through its C-terminal anchoring domain to prevent the release of cytochrome c after viral infection. This shows that the deletion of the F1L gene for NTV may be one of the more ideal transformation directions. Summary of the invention

[0008] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide a method for preparing a CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain, its vaccine strain and application. By inserting the C7L gene back while deleting the F1L gene, the modified NTV modified strain obtained the expected results. In the mouse toxicity experiment, it was proved that the toxicity of the NTV modified strain was close to that of NTV, and was also significantly lower than that of the Tiantan strain of vaccinia virus TTV. The humoral immunity and cellular immune response levels induced in mice by muscle immunization were higher than NTV, and were statistically significant. The NTV derivative strain is expected to become a candidate strain for the current smallpox / monkeypox vaccine in my country, and is undergoing pilot and clinical trial development.

[0009] Before describing the present invention, the terms used in this article are defined as follows:

[0010] The term CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) stands for "clustered regularly interspaced short palindromic repeats".

[0011] The term "CRISPR / Cas9" refers to a technology that makes specific DNA modifications to targeted genes.

[0012] The term "NTV-ΔF1L-C7L" refers to a recombinant vaccinia virus constructed based on the non-replicating vaccinia virus Tiantan strain, in which the F1L gene is deleted and the C7L gene is inserted back.

[0013] The term "NTV" refers to: Non-replicating vaccinia virus Tiantan strain.

[0014] The term "F1L gene" refers to: the first gene in the F region of the vaccinia virus genome.

[0015] The term "gRNA and Cas9 co-expression plasmid" refers to a plasmid used to simultaneously express gRNA and Cas9.

[0016] The term "pUC57" refers to the commercial plasmid pUC57, whose sequence is SEQ ID NO:14.

[0017] The term "pUC57-ΔF1L-LacZ-C7L" refers to a plasmid for constructing recombinant vaccinia virus NTV-ΔF1L-C7L constructed on the basis of the commercial plasmid pUC57.

[0018] The term "pX458del-616gRNA" refers to: a plasmid expressing 616gRNA and Cas9.

[0019] The term "pJSEDH6LacZ" refers to: a recombinant plasmid of the TK region of the vaccinia virus Tiantan strain.

[0020] The term "pUC57-ΔF1L-LacZ" refers to a plasmid constructed on the basis of the commercial plasmid pUC57 for recombination of the F1L region of vaccinia virus and insertion of the LacZ gene.

[0021] The term "LacZ" refers to the galactosidase gene.

[0022] The term "PCR" means: Polymerase Chain Reaction.

[0023] The term "VTT" refers to: Replication-competent vaccinia virus Tiantan strain.

[0024] The term "pMD18-T-C7L" refers to a plasmid obtained by ligating the C7L gene to the pMD18-T vector.

[0025] The term "pMD18-T vector" means: a special vector for efficient cloning of PCR products.

[0026] The term "pX458del" refers to the commercial plasmid pX458 with the nuclear localization gene deleted.

[0027] The term "pUC57-ΔF1L" refers to a plasmid for recombination of the vaccinia virus F1L region constructed based on the commercial plasmid pUC57.

[0028] The term "616gRNA" refers to: 616gRNA, the sequence of which is 5'-CACCGTGTTAGGGATGGCATTATC-3'.

[0029] The term "DNA oligonucleotide" refers to: generally refers to a linear polynucleotide fragment composed of 2 to 10 deoxynucleotide residues connected by phosphodiester bonds. The present invention refers to a DNA oligonucleotide formed by synthesizing the screened gRNA expression plasmid sequence targeting vaccinia virus, and undergoing phosphorylation modification and high-temperature deformation.

[0030] The term "BHK-21 cells" refers to: Syrian hamster kidney cells.

[0031] The term "E / L" refers to: promoter E / L, the sequence of which is 5'-CGAGCTTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTT-3'.

[0032] The term "P7.5" refers to: promoter P7.5, the sequence of which is 5'-CATCTATATACTATATAGTAATACCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATGTCGATAGCCATATGCCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACAGTGAC-3'.

[0033] The term "EcoRV" refers to: a type of restriction endonuclease.

[0034] The term "C7L" refers to the seventh gene in the C region of the vaccinia virus genome.

[0035] The term "C7L-F" refers to: C7L gene upstream primer 5'-CGGGATCCACCATGGGTATACAGCACGAATTC-3'.

[0036] The term "C7L-R" refers to: C7L gene downstream primer 5'-CGGGATCCCCGGGTTAATCCATGGACTCATAATC-3'.

[0037] The term "LoxP" refers to: Locus of X-over P1 originates from bacteriophage P1 and is a 34 bp DNA sequence.

[0038] The term "H6R" refers to: H6R promoter.

[0039] The term "ori" means: plasmid origin of replication.

[0040] The term "AmpR" refers to: the ampicillin gene on a commercial plasmid.

[0041] The term "U6 promoter" refers to: U6 promoter.

[0042] The term "gRNA scaffold" refers to: gRNA scaffold.

[0043] The term "CMV enhancer" refers to the enhancer of human cytomegalovirus (CMV).

[0044] The term "chicken β-actin promoter" refers to: chicken β-actin promoter.

[0045] The term "3×FLAG" means: 3 consecutive Flag tags.

[0046] The term "Cas9" refers to: Cas9 is an RNA-guided enzyme that cleaves foreign nucleic acids with sequences complementary to the RNA loaded into the enzyme during bacterial adaptive immunity.

[0047] The term "f1 ori" refers to: a single-stranded DNA replication origin on a plasmid.

[0048] The term "EGFP" means: Enhanced Green Fluorescent Protein.

[0049] The term "T2A" means: 2A peptides are short peptides (18-25 amino acids) derived from viruses. They are often called "self-cleaving" peptides and can produce multiple proteins from one transcription product.

[0050] The term "bGH poly(A) signal" refers to: bovine growth hormone polyadenylation signal.

[0051] The term "X-tremeGENE HP DNA" refers to: Transfection reagent from Roche.

[0052] The term "EDTA" means: ethylenediaminetetraacetic acid.

[0053] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain, the preparation method comprising the following steps:

[0054] (1) Construction of recombinant plasmid of vaccinia virus;

[0055] (2) Construction of a CRISPR / Cas9 system targeting vaccinia virus;

[0056] (3) Construction of recombinant vaccinia virus;

[0057] (4) Purification and characterization of vaccinia recombinant viruses; and

[0058] (5) Amplifying the vaccinia recombinant virus to obtain the CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain.

[0059] According to the preparation method of the first aspect of the present invention, the step (1) further comprises:

[0060] (A) preparing a first vector plasmid;

[0061] (B) digesting the first vector plasmid and the vaccinia virus TK region recombinant plasmid of step (A) respectively to obtain the selection marker LacZ gene and the first linearized plasmid, recovering and connecting the two to construct a second vector plasmid; and

[0062] (C) using primers C7L-F and C7L-R, and the replicative vaccinia virus Tiantan strain as a template, obtaining the PCR product C7L gene and connecting it to a vector to obtain a third vector plasmid, digesting the second vector plasmid and the third vector plasmid obtained in step (B) respectively to obtain the C7L gene and the second linearized plasmid, and connecting the two after recovery to construct the recombinant plasmid of the vaccinia virus;

[0063] Preferably, in the step (B), the vaccinia virus TK region recombinant plasmid is a vaccinia virus Tiantan strain TK region recombinant plasmid;

[0064] Preferably, the step (A) further comprises: selecting the upstream sequence of the F1L gene as the recombinant left arm and the downstream sequence of the F1L gene as the recombinant right arm according to the vaccinia virus NTV genome sequence, synthesizing the sequence and inserting it into a plasmid, thereby obtaining the first vector plasmid;

[0065] Preferably, the vaccinia virus early and late promoters are contained between the recombinant left arm and the recombinant right arm, and the sequence of the vaccinia virus early and late promoters is more preferably SEQ ID NO: 4 and / or SEQ ID NO: 13;

[0066] Preferably, the upstream sequence of the F1L gene is SEQ ID NO: 1 and / or SEQ ID NO: 3;

[0067] Preferably, the downstream sequence of the F1L gene is SEQ ID NO: 2; and / or

[0068] Preferably, the plasmid is pUC57, and its sequence is SEQ ID NO:14.

[0069] According to the preparation method of the first aspect of the present invention, in the step (C), before digestion, the steps further include: PCR contains restriction sites at both ends, and the second vector plasmid and the third vector plasmid obtained in the step (B) are water-bathed and digested separately with the restriction sites;

[0070] Preferably, the restriction site is BamHI;

[0071] Preferably, the temperature of the water bath is 25-50°C, more preferably 30-40°C, most preferably 37°C;

[0072] Preferably, the digestion time is 0.5 to 3 hours, more preferably 0.5 to 2 hours, most preferably 1 hour;

[0073] Preferably, in the step (C), the vector is pMD18-T;

[0074] Preferably, in step (C), the sequence of the primer C7L-F is SEQ ID NO: 5, and the sequence of the primer C7L-R is SEQ ID NO: 6; and / or

[0075] Preferably, in the step (C), the nucleotide sequence of the recombinant plasmid of the vaccinia virus is as shown in SEQ ID NO:7.

[0076] According to the preparation method of the first aspect of the present invention, in the step (2), the CRISPR / Cas9 system targeting vaccinia virus comprises: a plasmid for co-expression of gRNA targeting vaccinia virus and Cas9 gene;

[0077] Preferably, the plasmid for co-expression of the gRNA and Cas9 gene targeting vaccinia virus includes a plasmid for co-expression of the gRNA and Cas9 gene having the sequence shown in SEQ ID NO: 10.

[0078] According to the preparation method of the first aspect of the present invention, in the step (2), the preparation method of the CRISPR / Cas9 system targeting vaccinia virus comprises:

[0079] (D) Screening of gRNA expression plasmids targeting vaccinia virus;

[0080] (E) synthesizing the gRNA expression plasmid sequence screened in step (D), and forming a DNA oligonucleotide after phosphorylation modification and high temperature denaturation; and

[0081] (F) digesting and recovering the DNA oligonucleotide prepared in step (E) and the gRNA expression plasmid and Cas9 gene expression vector plasmid screened in step (D) to obtain the gRNA sequence and the linearized Cas9 gene expression vector plasmid, and connecting the two after recovery to construct the CRISPR / Cas9 system targeting vaccinia virus;

[0082] Preferably, in the step (D), the gRNA targeting vaccinia virus comprises an expression plasmid expressing a gRNA having a sequence shown in SEQ ID NO: 8;

[0083] Preferably, in the step (E), the DNA oligonucleotide comprises a DNA oligonucleotide expressing a sequence shown in SEQ ID NO: 12;

[0084] Preferably, in the step (F), the nucleotide sequence of the Cas9 gene expression vector plasmid is as shown in SEQ ID NO:9.

[0085] According to the preparation method of the first aspect of the present invention, in step (3), the operation of constructing the vaccinia recombinant virus further comprises the following steps:

[0086] (a) growing cells;

[0087] (b) transfecting the CRISPR / Cas9 system targeting vaccinia virus using a transfection reagent and inoculating non-replicating vaccinia virus; and

[0088] (c) transfecting a recombinant plasmid of vaccinia virus and inoculating a non-replicating vaccinia virus, harvesting cells and supernatant, and freezing to obtain the recombinant vaccinia virus;

[0089] Preferably, the cell in step (a) is BHK-21;

[0090] Preferably, in step (b), the transfection reagent is X-tremeGENE HP DNA;

[0091] Preferably, in step (c), the nucleotide sequence of the recombinant region of the vaccinia recombinant virus is as shown in SEQ ID NO: 11; and / or

[0092] Preferably, in step (b) and step (c), the non-replicating vaccinia virus is NTV.

[0093] According to the preparation method of the first aspect of the present invention, the step (4) further comprises: thawing the vaccinia recombinant virus and inoculating it into BHK-21 cells for purification and identification;

[0094] Preferably, the number of thawing is 2 to 5 times, more preferably 2 to 4 times, and most preferably 3 times;

[0095] Preferably, the BHK-21 cells are BHK-21 cells grown to 80-95%, more preferably BHK-21 cells grown to 85-95%, and most preferably BHK-21 cells grown to 90%; and / or

[0096] Preferably, the identification includes: observing enzyme cleavage bands and analyzing with an infrared fluorescence scanning imaging system.

[0097] According to the preparation method of the first aspect of the present invention, the step (5) further comprises the following steps:

[0098] (d) inoculating the vaccinia recombinant virus into cells, centrifuging after culturing, discarding the supernatant, collecting the cell precipitate, washing and centrifuging the cell precipitate, and sonicating after thawing;

[0099] (e) adding pancreatin to the sonicated vaccinia recombinant virus solution, incubating and digesting, and then centrifuging; the pancreatin is preferably EDTA-free pancreatin;

[0100] (f) adding sucrose solution to separate the layers, centrifuging, discarding the supernatant, resuspending the precipitate, and storing it for later use;

[0101] Preferably, in step (d), the ultrasonic power is 50-70W, the time is 3-5s, the interval is 6-9s, and the number is 25-35 times, and the most preferred ultrasonic power is 60W, the time is 4s, the interval is 8s, and the number is 30 times; and / or

[0102] Preferably, in step (f), the storage temperature is -90 to -50°C, more preferably -80 to -60°C, most preferably -70°C.

[0104] The second aspect of the present invention provides a vaccinia virus genetically modified vaccine strain, which is prepared by the preparation method described in the first aspect.

[0105] The third aspect of the present invention provides the use of the vaccinia virus gene-modified vaccine strain described in the second aspect in the preparation of monkeypox vaccine products or smallpox vaccine replacement products.

[0106] Some existing technologies are to construct non-replicating vaccinia virus NTV based on vaccinia virus TTV. Construction of F1L region recombination system of vaccinia virus Tiantan strain. Construction of recombinant plasmid, construction of recombinant vaccinia virus NTV-ΔF1L-C7L and evaluation of immune effect are not disclosed.

[0107] The present invention utilizes homologous recombination to construct a derivative strain of NTV, NTV-ΔF1L-C7L, which makes up for the original NTV's insufficient immunogenicity and makes the invention more potential as a smallpox or monkeypox vaccine.

[0108] In this experiment, CRISPR / Cas9 editing technology was used to first construct the recombinant plasmid pUC57-ΔF1L-LacZ-C7L and the gRNA and Cas9 co-expression plasmid pX458del-616gRNA. Using the principle of homologous recombination, the non-replicating vaccinia virus NTV underwent homologous recombination with the recombinant plasmid pUC57-ΔF1L-LacZ-C7L under the mediation of CRISPR / Cas9 during the intracellular replication process. While the F1L gene was deleted in the NTV genome, the host range gene C7L and the selection marker gene LacZ were inserted back. After purification and identification of the recombinant virus, the recombinant virus NTV-ΔF1L-C7L expressing C7 protein and lacking F1 protein was screened and amplified. The immunogenicity of the recombinant virus NTV-ΔF1L-C7L was evaluated by animal immunization experiments.

[0109] The recombinant vaccinia virus was constructed by using a CRISPR / Cas9-based homologous recombination method to obtain the virus quickly. The recombinant virus can be used as a candidate vaccine strain for the next generation of monkeypox vaccine or smallpox vaccine.

[0110] The purpose of the present invention is to construct a gene-modified vaccinia virus vaccine strain with low toxicity and strong immune effect, which can be used as a candidate vaccine strain for alternative products for the prevention of infection with existing smallpox vaccines in my country, as well as a preventive vaccine for monkeypox virus infection.

[0111] The nucleotide sequences of the present invention are shown in SEQ ID NO.1-14:

[0112] SEQ ID NO: 1: 5'-GGATAGGGGTCTTCATAACGCAT-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 1 shall be based on the sequence described herein in the specification).

[0113] SEQ ID NO: 2: 5'-GATAGGAGGTGGTGTAATAGACGAAG-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 2 shall be based on the sequence described herein in the specification).

[0114] SEQ ID NO: 3: 5'-ATGAGGATGCTGTTTTTTACTTTGTGG-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 3 shall be based on the sequence described herein in the specification).

[0115] SEQ ID NO: 4: 5'-GGGTGGGTTTGGAATTAGTG-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 4 shall be based on the sequence described herein in the specification).

[0116] SEQ ID NO: 5: 5'-CGGGATCCACCATGGGTATACAGCACGAATTC-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 5 shall be based on the sequence described herein in the specification).

[0117] SEQ ID NO:6: 5'-CGGGATCCCCGGGTTAATCCATGGACTCATAATC-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO.6 shall be based on the sequence described herein in the specification).

[0118] SEQ ID NO:7: 5'-AATTCGAGCTCTCATTGTCGATAGATCTAGGCCTCGAGCTTATTTATA TTCCAAAAAAAAAAAATAAAATTTCAATTTTTAAGCTTCATCTATATACTATATAGTAATACCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATGTCGATAGCCATATGCCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAAGT

[0119] AGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAG

[0120] AACAGTGACGGATCCACCATGGGTATACAGCACGAATTCGACATCATT

[0121] ATTAATGGAGATATCGCGTTGAGAAATTTACAGTTACATAAAGGGGATA

[0122] ACTACGGATGCAAACTAAAAATTATTTCGAATGATTACAAGAAATTAA

[0123] AGTTTAGATTCATTATACGCCCAGATTGGTCGGAAATCGACGAGGTCA

[0124] AAGGATTAACCGTATTTGCAAACAACTATGCGGTGAAAGTTAATAAGG

[0125] TAGATGACACGTTCTATTACGTAATATATGAGGCTGTAATACATCTGTAT

[0126] AACAAAAAAACAGAGATATTGATTTATTCTGATGATGAGAACGAGCTC

[0127] TTCAAACACTATTACCCATACATCAGTCTAAATATGATTAGTAAAAAGT

[0128] ATAAAGTTAAAGAAGAGAACTACTCATCCCCGTATATAGAACATCCGTT

[0129] AATCCCGTATAGAGATTATGAGTCCATGGATTAACCCGGGGATCCCCGG

[0130] GATAACTTCGTATAATGTATGCTATACGAAGTTATTTATTTTTGACACCA

[0131] GACCAACTGGTAATGGTAGCGACCGGCGCTCAGCTGTAATTCCGCCGA

[0132] TACTGACGGGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACC

[0133] GTCGATATTCAGCCATGTGCCTTCTTCCGCGTGCAGCAGATGGCGATG

[0134] GCTGGTTTCCATCAGTTGCTGTTGACTGTAGCGGCTGATGTTGAACTG

[0135] GAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGCGTCCC

[0136] GCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTC

[0137] TGACAATGGCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGT

[0138] CGGGATAGTTTTCTTGCGGCCCTAATCCGAGCCAGTTTACCCGCTCTGC

[0139] TACCTGCGCCAGCTGGCAGTTCAGGCCAATCCGCGCCGGATGCGGTGT

[0140] ATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCACTACCA

[0141] TCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCT

[0142] GCCACGCGTGAGCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTG

[0143] CCGTGCACTGCAACAACGCTGCTTCGGCCTGGTAATGGCCCGCCGCCT

[0144] TCCAGCGTTCGACCCAGGCGTTAGGGTCAATGCGGGTCGCTTCACTTA

[0145] CGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCGCGCAGCG

[0146] GCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGC

[0147] CTGACTGGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAA

[0148] AATCCATTTCGCTGGTGGTCAGATGCGGGATGGCGTGGGACGCGGCG

[0149] GGGAGCGTCACACTGAGGTTTTCCGCCAGACGCCACTGCTGCCAGGC

[0150] GCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCACTAC

[0151] GCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTC

[0152] AGGCAGTTCAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCAC

[0153] TTCACCGCTTGCCAGCGGCTTACCATCCAGCGCCACCATCCAGTGCAG

[0154] GAGCTCGTTATCGCTATGACGGAACAGGTATTCGCTGGTCACTTCGAT

[0155] GGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGTTTTG

[0156] CTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCG

[0157] TTCATACAGAACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCG

[0158] TAAGCCGACCACGGGTTGCCGTTTTCATCATATTTAATCAGCGACTGAT

[0159] CCACCCAGTCCCAGACGAAGCCGCCCTGTAAACGGGGATACTGACGA

[0160] AACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATCGCG

[0161] TGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGA

[0162] AAGCCATTTTTTGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTC

[0163] TTCATCCACGCGCGCGTACATCGGGCAAATAATATCGGTGGCCGTGGT

[0164] GTCGGCTCCGCCGCCTTCATACTGCACCGGGCGGGAAGGATCGACAG

[0165] ATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATT

[0166] CATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCT

[0167] GCACCATTCGCGTTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGAT

[0168] CATCGGTCAGACGATTCATTGGCACCATGCCGTGGGTTTCAATATTGGC

[0169] TTCATCCACCACATACAGGCCGTAGCGGTCGCACAGCGTGTACCACAG

[0170] CGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTCTG

[0171] CTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGA

[0172] CCATGCAGAGGATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAAC

[0173] GGCTTGCCGTTCAGCAGCAGCAGACCATTTTCAATCCGCACCTCGCGG

[0174] AAACCGACATCGCAGGCTTCTGCTTCAATCAGCGTGCCGTCGGCGGTG

[0175] TGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTCCAC

[0176] AGTTTCGGGTTTTCGACGTTCAGACGTAGTGTGACGCGATCGGCATAA

[0177] CCACCACGCTCATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTG

[0178] GCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAG

[0179] TCACGCAACTCGCCGCACATCTGAACTTCAGCCTCCAGTACAGCGCG

[0180] GCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTTG

[0181] TGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCAT

[0182] CCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTCCAGCGCAG

[0183] CACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTCAG

[0184] GTCAAATTCAGACGGCAAACGACTGTCCTGGCCGTAACCGACCCAGC

[0185] GCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATAA

[0186] TTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAG

[0187] CGAGTAACAACCCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGA

[0188] CCGTAATGGGATAGGTCACGTTGGTGTAGATGGGCGCATCGTAACCGT

[0189] GCATCTGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAGGAAGA

[0190] TCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCA

[0191] GGCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGC

[0192] GATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGAT

[0193] GTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCAC

[0194] GACGTTGTAAAACGACGGGATCGCGCTTGAGCAGCTCCTTGCTGGTG

[0195] TCCAGACCAATGCCTCCCAGACCGGCAACGAAAATCACGTTCTTGTTG

[0196] GTCAAAGTAAACGACATGGTGACTTCTTTTTTGCTTTAGCAGGCTCTTT

[0197] CGATCCCCGATCTATTACGATACAAACTTAACGGATATCGCGATAATGA

[0198] AATAATTTATGATTATTTCTCGCTTTCAATTTAACACAACCCTCAAGAA

[0199] CCTTTGTATTTATTTTCATTTTTTAAGTATAGAATAAAGAATCTATAAAA

[0200] ACTAAAAAAATTATACATTATAAACCAATTTCCTAGTTGTTTGTAAATAA

[0201] CTTCGTATAATGTATGCTATACGAAGTTATCCCGGGCTATTGTTTATTATC

[0202] TAAGTCCTGTTGATCCAAACCCTTGATCTCCTCTATCTGTACTATCTAGA

[0203] GATTGTACTTCTTCCAGTTCTGGATAATATATACGTTGATAGATTAGCTG

[0204] AGCTATTCTATCTCCAGTATTTACATTAAACGTACATTTTCCATTATTAAT

[0205] AAGAATGACTCCTATGTTTCCCCTATAATCTTCGTCTATTACACCACCTC

[0206] CTATATCAATGCCTTTTAGTGACAGACCAGACCTAGGAGCTATTCTACC

[0207] ATAGCAAATCTTAGGCATGGACATACTAATATCTGTCTTAATTAACTGTC

[0208] TTTCTCCTGGAGGGATAGTATAATCGTAAGCGCTATACAAATCATATCC

[0209] GGCAGCACCCGGCGATTGCCTAGTAGGAGATTTAGCTCTGTTAGTTTC

[0210] CTTAACAAATCTAACTGGTGAGTTAATATTCATGTTGAACATAAAACTA

[0211] ATATTTTATTTCAAAATTATTTACCATCCCATATATTCCATGAATAAGTGT

[0212] GATGATTGTACACTTCTATAGTATCTATATACGATTCACGATAAAATCCT

[0213] CCTATCAATAGCAGTTTATTATCCACTATGATCAATTCTGGATTATCCCT

[0214] CGGATAAATAGGATCATCTATCAGAGTCCATGTATTGCTGGATTCACAA

[0215] TAAAATTCCGCATTTCTACCAACCAAGAATAACCTTCTACCGAACACTA

[0216] ACGCGCATGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCG

[0217] TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAA

[0218] TTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGT

[0219] GCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCC

[0220] GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC

[0221] CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC

[0222] TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTA

[0223] TCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGAT

[0224] AACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA

[0225] ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCC

[0226] CTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC

[0227] CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTC

[0228] GTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC

[0229] TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT

[0230] ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG

[0231] AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC

[0232] TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC

[0233] ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA

[0234] GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATT

[0235] TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG

[0236] TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT

[0237] TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG

[0238] ATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT

[0239] CACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTA

[0240] GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATG

[0241] AGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTA

[0242] TCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT

[0243] CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGC

[0244] TGCAATGATACCGCGAGACCCAGCCTCACCGGCTCCAGATTTATCAGC

[0245] AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA

[0246] ACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAG

[0247] TAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC

[0248] AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCC

[0249] GGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCCATGTTGTGCAAA

[0250] AAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG

[0251] GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA

[0252] CTGTCATGCCATCCGTAAGATGCTTTCTGTGACTGGTGAGTACTCAAAC

[0253] CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC

[0254] GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT

[0255] GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT

[0256] ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTG

[0257] ATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC

[0258] AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA

[0259] TGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCA

[0260] GGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT

[0261] AAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA

[0262] CGTCCAGTTTGCACTCATGATTCACGTTATATAACTATCAATATTAACAG

[0263] TTCGTTTGATGATCATATTATTTTTATGTTTTATTGATAATTGTAAAAACA

[0264] TACAATTAAATCAATATAGAGGAAGGAGACGGCTACTGTCTTTTGTGA

[0265] GATAGTCATGGCGACTAAATTAGATTATGAGGATGCTGTTTTTTACTTT

[0266] GTGGATGATGATAAAATATGTAGTCGCGACTCCATCATCGATCTAATAG

[0267] ATGAATATATTACGTGGAGAAATCATGTTATAGTGTTTAACAAAGATATT

[0268] ACCAGTTGTGGAAGACTGTACAAGGAATTGATGAAGTTCGATGATGTC

[0269] GCTATACGGTACTATGGTATTGATAAAATTAATGAGATTGTCGAAGCTAT

[0270] GAGCGAAGGAGACCACTACATCAATTTTACAAAAGTCCATGATCAGGA

[0271] AAGTTTATTCGCTACCATAGGAATATGTGCTAAAATCACTGAACATTGG

[0272] GGATACAAAAAGATTTCAGAATCTAGATTCCAATCATTGGGAAACATT

[0273] ACAGATCTGATGACCGACGATAATATAAACATCTTGATACTTTTTCTAG

[0274] AAAAAAAATTGAATTGATGATATAGGGGTCTTCATAACGCATAATTATTACGTTAGCATTCTATATCCGTGTTAAAAAAAAG-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 7 shall be based on the sequence described herein in the specification).

[0275] SEQ ID NO: 8: 5'-CACCGTGTTAGGGATGGCATTATC-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 8 shall be based on the sequence described herein in the specification).

[0276]

[0277] CGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCC

[0278] CAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA

[0279] CCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATG

[0280] AGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGG

[0281] ACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAA

[0282] GAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCC

[0283] GGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTG

[0284] CTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGA

[0285] GGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAG

[0286] AGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACG

[0287] ACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGC

[0288] AGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGG

[0289] CAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAA

[0290] ACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACA

[0291] TCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCAC

[0292] ATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAG

[0293] ACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACA

[0294] AGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACC

[0295] CAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAG

[0296] AGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTG

[0297] GAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCA

[0298] GAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGC

[0299] TGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGG

[0300] ACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCG

[0301] GGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATG

[0302] AAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAG

[0303] AAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAA

[0304] CTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCA

[0305] GATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTA

[0306] AGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACC

[0307] CTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTAC

[0308] AAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCT

[0309] GAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGG

[0310] AAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAG

[0311] ATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTA

[0312] CTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTG

[0313] GCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA

[0314] AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC

[0315] GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAG

[0316] GTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAA

[0317] CAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGT

[0318] ACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGG

[0319] CCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGA

[0320] GCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATC

[0321] CCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGAC

[0322] CTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGC

[0323] CGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACG

[0324] AACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCC

[0325] ACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAG

[0326] CTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCA

[0327] GATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGA

[0328] CAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAG

[0329] AGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAG

[0330] CCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGT

[0331] ACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGC

[0332] ATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC-3’(Since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO.9 shall be subject to the sequence recorded herein in the specification).

[0333] SEQ ID NO:10: 5’-GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAG GCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA

[0334] CACCGTGTTAGGGATGGCATTATCGTTTTAGAGCTAGAAATAGCAAGTT

[0335] AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG

[0336] GTGCTTTTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT

[0337] CCGTTTTTAGCGCGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGT

[0338] ACCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA

[0339] CGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCA

[0340] TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT

[0341] ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC

[0342] GGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGAC

[0343] TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT

[0344] CGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTC

[0345] CCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGAT

[0346] GGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCG

[0347] GGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC

[0348] CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGC

[0349] GGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT

[0350] CGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCG

[0351] CCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCG

[0352] GGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGT

[0353] AAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCT

[0354] GGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGACCGGTGCCACC

[0355] ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGAT

[0356] TACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGT

[0357] CGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCC

[0358] TGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAG

[0359] TACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCG

[0360] GCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCG

[0361] GCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAG

[0362] ATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAG

[0363] CAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAG

[0364] AGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATC

[0365] TTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCAC

[0366] CATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCG

[0367] ACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGG

[0368] GCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTG

[0369] GACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA

[0370] GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGT

[0371] CTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAG

[0372] CTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCT

[0373] GAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCG

[0374] AGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTG

[0375] GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCT

[0376] GGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGA

[0377] GAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATC

[0378] AAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCT

[0379] CGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACC

[0380] AGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAG

[0381] GAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG

[0382] CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGG

[0383] AAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCT

[0384] GGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATT

[0385] CCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCA

[0386] TCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCT

[0387] GGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAG

[0388] GAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGAT

[0389] GACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGC

[0390] ACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAG

[0391] TGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGC

[0392] GAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAA

[0393] AGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGT

[0394] GCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCT

[0395] CCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACT

[0396] TCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTG

[0397] ACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAA

[0398] AACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC

[0399] GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAAC

[0400] GGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAA

[0401] GTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACG

[0402] ACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGC

[0403] CAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCC

[0404] CGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGC

[0405] TCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAA

[0406] ATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCC

[0407] GCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAG

[0408] CCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACG

[0409] AGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGG

[0410] ACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATA

[0411] TCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTG

[0412] CTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCT

[0413] CCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTG

[0414] AACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGC

[0415] CGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGA

[0416] GACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATC

[0417] CTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGAT

[0418] CCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATT

[0419] TCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACC

[0420] ACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTG

[0421] ATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTA

[0422] CAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAA

[0423] TCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACT

[0424] TTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG

[0425] CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAA

[0426] GGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAG

[0427] TGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAA

[0428] GAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAA

[0429] GAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG

[0430] TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCC

[0431] AAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGA

[0432] AAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGG

[0433] GCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC

[0434] TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGC

[0435] CGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATG

[0436] TGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCC

[0437] CCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCAC

[0438] TACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGT

[0439] GATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAA

[0440] GCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCT

[0441] GTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGA

[0442] CACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGG

[0443] ACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGG

[0444] ATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAA

[0445] AAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAG

[0446] GGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGG

[0447] CCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCC

[0448] TGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCC

[0449] GGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT

[0450] CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGAC

[0451] CACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACAT

[0452] GAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

[0453] AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGC

[0454] GCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCT

[0455] GAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC

[0456] TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGC

[0457] AGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAG

[0458] GACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT

[0459] CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCC

[0460] AGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC

[0461] CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA

[0462] GCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTG

[0463] CCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT

[0464] TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG

[0465] AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGG

[0466] GGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGG

[0467] CATGCTGGGGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCAC

[0468] TCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG

[0469] TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA

[0470] GCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACG

[0471] CATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACG

[0472] CGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC

[0473] AGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCT

[0474] TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC

[0475] TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT

[0476] CGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATC

[0477] GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT

[0478] AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCG

[0479] GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTT

[0480] AAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT

[0481] ATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGAT

[0482] GCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGC

[0483] GCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGT

[0484] GACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

[0485] GAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGT

[0486] TAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGG

[0487] GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA

[0488] ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT

[0489] TGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATT

[0490] CCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC

[0491] TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT

[0492] TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGC

[0493] CCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGT

[0494] GGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGC

[0495] CGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA

[0496] GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCT

[0497] GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG

[0498] ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA

[0499] TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT

[0500] ACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA

[0501] CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC

[0502] AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC

[0503] TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

[0504] CGGTGAGCGTGGAAGCCGCGGTATCATTGCAGCACTGGGGCCAGATG

[0505] GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA

[0506] CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA

[0507] TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT

[0508] GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTT

[0509] TTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTG

[0510] AGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

[0511] TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC

[0512] AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA

[0513] GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGT

[0514] GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC

[0515] ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGAT

[0516] AAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAG

[0517] GCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT

[0518] GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT

[0519] GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC

[0520] GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCA

[0521] GGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC

[0522] TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA

[0523] TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGC

[0524] TGGCCTTTTGCTCACATGT-3’(Since the WIPO Sequence cannot recognize 5'- and -3',

[0525] SEQ ID NO.10 shall be subject to the sequence recorded herein in the specification).

[0526]

[0527] ATGTATGCTATACGAAGTTATTTATTTTTGACACCAGACCAACTGGTAA

[0528] TGGTAGCGACCGGCGCTCAGCTGTAATTCCGCCGATACTGACGGGCTC

[0529] CAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGC

[0530] CATGTGCCTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATC

[0531] AGTTGCTGTTGACTGTAGCGGCTGATGTTGAACTGGAAGTCGCCGCGC

[0532] CACTGGTGTGGGCCATAATTCAATTCGCGCGTCCCGCAGCGCAGACCG

[0533] TTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAATGGCAGA

[0534] TCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTC

[0535] TTGCGGCCCTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAG

[0536] CTGGCAGTTCAGGCCAATCCGCGCCGGATGCGGTGTATCGCTCGCCAC

[0537] TTCAACATCAACGGTAATCGCCATTTGACCACTACCATCAATCCGGTAG

[0538] GTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCACGCGTGA

[0539] GCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGC

[0540] AACAACGCTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCG

[0541] ACCCAGGCGTTAGGGTCAATGCGGGTCGCTTCACTTACGCCAATGTCG

[0542] TTATCCAGCGGTGCACGGGTGAACTGATCGCGCAGCGGCGTCAGCAG

[0543] TTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGACTGGCG

[0544] GTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCG

[0545] CTGGTGGTCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTCAC

[0546] ACTGAGGTTTTCCGCCAGACGCCACTGCTGCCAGGCGCTGATGTGCCC

[0547] GGCTTCTGACCATGCGGTCGCGTTCGGTTGCACTACGCGTACTGTGAG

[0548] CCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGTTCAAT

[0549] CAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGC

[0550] CAGCGGCTTACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATC

[0551] GCTATGACGGAACAGGTATTCGCTGGTCACTTCGATGGTTTGCCCGGA

[0552] TAAACGGAACTGGAAAAACTGCTGCTGGTGTTTTGCTTCCGTCAGCG

[0553] CTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGAAC

[0554] TGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCAC

[0555] GGGTTGCCGTTTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCC

[0556] AGACGAAGCCGCCCTGTAAACGGGGATACTGACGAAACGCCTGCCAG

[0557] TATTTAGCGAAACCGCCAAGACTGTTACCCATCGCGTGGGCGTATTCG

[0558] CAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTTTTG

[0559] ATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGC

[0560] GCGTACATCGGGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCG

[0561] CCTTCATACTGCACCGGGCGGGAAGGATCGACAGATTTGATCCAGCGA

[0562] TACAGCGCGTCGTGATTAGCGCCGTGGCCTGATTCATTCCCCAGCGAC

[0563] CAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGCGTT

[0564] ACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGA

[0565] TTCATTGGCACCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACAT

[0566] ACAGGCCGTAGCGGTCGCACAGCGTGTACCACAGCGGATGGTTCGGA

[0567] TAATGCGAACAGCGCACGGCGTTAAAGTTGTTCTGCTTCATCAGCAGG

[0568] ATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGAGGAT

[0569] GATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCA

[0570] GCAGCAGCAGACCATTTTCAATCCGCACCTCGCGGAAACCGACATCG

[0571] CAGGCTTCTGCTTCAATCAGCGTGCCGTCGGCGGTGTGCAGTTCAACC

[0572] ACCGCACGATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTT

[0573] TCGACGTTCAGACGTAGTGTGACGCGATCGGCATAACCACCACGCTCA

[0574] TCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTT

[0575] TCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCG

[0576] CCGCACATCTGAACTTCAGCCTCCAGTACAGCGCGGCTGAAATCATCA

[0577] TTAAAGCGAGTGGCAACATGGAAATCGCTGATTTGTGTAGTCGGTTTA

[0578] TGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATATCC

[0579] TGATCTTCCAGATAACTGCCGTCACTCCAGCGCAGCACCATCACCGCG

[0580] AGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGAC

[0581] GGCAAACGACTGTCCTGGCCGTAACCGACCCAGCGCCCGTTGCACCA

[0582] CAGATGAAACGCCGAGTTAACGCCATCAAAAATAATTCGCGTCTGGCC

[0583] TTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAACC

[0584] CGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATA

[0585] GGTCACGTTGGTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTT

[0586] TGAGGGGACGACGACAGTATCGGCCTCAGGAAGATCGCACTCCAGCC

[0587] AGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCAT

[0588] TCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGC

[0589] CTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCG

[0590]

[0591] SEQ ID NO: 12: Its forward sequence is: 5'-CACCGTGTTAGGGATGGCATTATC-3' (Since WIPOSEQUENCE cannot recognize 5'- and -3', SEQ ID NO. 12 is based on the sequence recorded below in the brackets. Specifically, SEQ ID NO: 12 is a double-stranded DNA with sticky ends, and its sequence is:

[0592] 5'-CACCGTGTTAGGGATGGCATTATC-3'

[0593] 3'-CACAATCCCTACCGTAATAGCAAA-5'

[0594] Among them, CACC and CAAA are the sticky ends of double-stranded DNA, synthesized by Nanjing GenScript Biotech Co., Ltd. The two chains are heat-denatured and annealed to form double-stranded DNA).

[0595] SEQ ID NO: 13: 5'-CGAGCTTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTT-3' (since WIPO Sequence cannot recognize 5'- and -3', SEQ ID NO. 13 shall be based on the sequence described herein in the specification).

[0596]

[0597] The CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain of the present invention may have but is not limited to the following beneficial effects:

[0598] 1. The recombinant virus NTV-ΔF1L-C7L can be well replicated and amplified in chicken embryo cells, ensuring the production of the virus. Secondly, the virus can induce effective humoral immunity and cellular immunity in mice, indicating that the recombinant virus has the potential to be a replacement product for monkeypox vaccine or smallpox vaccine.

[0599] 2. The modified NTV strain obtained the expected results. The mouse virulence experiment proved that the virulence of the modified NTV strain was close to that of NTV, and was significantly lower than that of the Tiantan strain of vaccinia virus TTV. The humoral and cellular immune responses induced in mice by muscle immunization were higher than those of NTV, and were statistically significant. The NTV derivative strain is expected to become a candidate strain for the current smallpox / monkeypox vaccine in my country, and is undergoing pilot and clinical trial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0600] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0601] Figure 1 A schematic diagram of the structure of the recombinant plasmid pUC57-ΔF1L-LacZ-C7L is shown.

[0602] Figure 2 A schematic diagram of the structure of the gRNA and Cas9 co-expression plasmid pX458del-616gRNA is shown.

[0603] Figure 3 A schematic diagram of the structure of the NTV-ΔF1L-C7L recombination region of the recombinant vaccinia virus is shown.

[0604] Figure 4 The PCR and Western blot results of the recombinant virus NTV-ΔF1L-C7L are shown; wherein, Figure 4 A shows the PCR identification results of the recombinant virus; Figure 4 B shows the Western blot identification results of the recombinant virus.

[0605] Figure 5 The weight change curve and survival curve of mice after intranasal inoculation are shown; Figure 5 A shows a dose of 10 6 Mouse weight change curve at PFU; Figure 5 B shows a dose of 10 7 Mouse weight change curve at PFU; Figure 5 C shows a dose of 5 × 107 Mouse weight change curve at PFU; Figure 5 D shows a dose of 10 6 Survival curve of mice at PFU; Figure 5 E shows a dose of 10 7 Survival curve of mice at PFU; Figure 5 F shows a dose of 5 × 10 7 Survival curve of mice at PFU.

[0606] FIG6 shows the cellular immune response induced by vaccinia virus; wherein, Fig. 6A ELISPOT results after E3 peptide stimulation are shown; Figure 6B Shown are the ELISPOT results after F2 peptide stimulation.

[0607] FIG. 7 shows the humoral immune response induced by vaccinia virus; wherein, Fig. 7A Vaccinia virus-specific IgG antibody titers are shown; Figure 7B Vaccinia virus-specific IgG1 and IgG2a antibody titers are shown; Figure 7C The ratio of IgG2a to IgG1 antibody titers is shown. DETAILED DESCRIPTION

[0608] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0609] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0610] The reagents and instruments used in the following examples are as follows:

[0611] Reagents:

[0612] PrimeSTAR Max DNAPolymerase, Taq DNA Polymerase, 18-TVector, λ-HindⅢmarker, DL5000 marker were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; Tiangen small and large extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; commonly used restriction endonucleases, T4 DNA ligase, and T4 PNK enzyme were purchased from NEB; prestained protein markers and GeneJET gel recovery kit were purchased from Thermo Fisher Scientific (China) Co., Ltd.; transfection reagent X-tremeGEN HP DNA Transfection Reagent was purchased from Roche Pharmaceuticals Co., Ltd.; Qiagen blood / tissue genomic DNA extraction kit was purchased from QIAGEN;

[0613] DMEM medium, Opti-MEM medium, Fetal Bovine Serum (FBS), 0.25% trypsin (containing EDTA) and 0.25% trypsin (without EDTA) were all purchased from Gibco, USA; 2×DMEM was purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.

[0614] instrument:

[0615] CO 2 The cell culture incubator was purchased from Singapore Yisi Technology Co., Ltd., model: CCL-170B-8.

[0616] Optical microscope, purchased from Nikon, model: ECLIPSE TS100-F.

[0617] The constant temperature water bath was purchased from Shanghai Senxin Experimental Instrument Co., Ltd., model: DK-420.

[0618] Cole-Parmer ultrasonic instrument, purchased from Cole-Parmer Instrument (Shanghai) Co., Ltd., model: CPX 130.

[0619] Eppendorf 5810R centrifuge, purchased from Eppendorf, Germany, model: 5810R.

[0620] Ultracentrifuge, purchased from Beckman Coulter Trading (China) Co., Ltd., model: L-80XP.

[0621] The biological safety cabinet was purchased from Thermo Fisher Scientific, model: 1354.

[0622] Bio-Rad Trans-Blot Cell semi-dry transfer instrument was purchased from Bio-Rad Biomedical Products (Shanghai) Co., Ltd., model: 221BR.

[0623] Bio-Rad gel imaging instrument was purchased from Bio-Rad Biomedical Products (Shanghai) Co., Ltd., model: GELDOCXR.

[0624] Odyssey infrared fluorescence scanning imaging system, purchased from LI-COR, model: Odyssey.

[0625] In the following examples, the detection primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0626] Plasmid pUC57 was synthesized by Nanjing GenScript Biotech Co., Ltd.

[0627] Experimental animals: BALB / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0628] The bio-genetic engineering operation techniques involved in the present invention (such as plasmid extraction, enzyme digestion, fragment recovery, nucleic acid fragment and plasmid vector connection reaction, cloning and screening, etc., have been described in detail in the Molecular Cloning Guide (Second Edition).

[0629] Example 1 Design and synthesis of vaccinia virus recombinant plasmid pUC57-ΔF1L-LacZ-C7L

[0630] According to the NTV genome sequence, the 666bp upstream of the F1L gene was selected as the recombinant left arm, and the 719bp downstream of the F1L gene was selected as the recombinant right arm. The vaccinia virus early and late promoters P7.5 and E / L were contained between the left and right arms. The sequence was sent to Nanjing GenScript Biotech Co., Ltd. for synthesis and inserted into the plasmid pUC57, and the plasmid was named pUC57-△F1L.

[0631] The recombinant plasmid pJSEDH6LacZ of the TK region of the Tiantan strain of vaccinia virus and the synthetic plasmid pUC57-△F1L were digested with XmaⅠ in a 37℃ water bath for 1 hour to obtain the selection marker LacZ gene and the linearized plasmid pUC57-△F1L. After recovery by the kit, the two were connected to construct the plasmid pUC57-△F1L-LacZ.

[0632] Using primers C7L-F and C7L-R and replicative vaccinia virus Tiantan strain VTT as template, the PCR product C7L gene was obtained and connected to the pMD18-T vector, named pMD18-T-C7L. The PCR product contains BamHI restriction sites at both ends. The plasmids pUC57-△F1L-LacZ and pMD18-T-C7L were digested with BamHI-HF in a 37℃ water bath for 1 hour to obtain the C7L gene and the linearized plasmid pUC57-△F1L-LacZ. After recovery by the kit, the two were connected to construct the plasmid pUC57-△F1L-LacZ-C7L.

[0633] The left arm sequence is shown in SEQ ID NO:1 and SEQ ID NO:3.

[0634] The right arm sequence is shown in SEQ ID NO:2.

[0635] The sequence of the vaccinia virus early and late promoter P7.5 is shown in SEQ ID NO:4.

[0636] The vaccinia virus early and late promoter E / L sequence is shown in SEQ ID NO:13.

[0637] The sequence of primer C7L-F is shown in SEQ ID NO:5.

[0638] The sequence of primer C7L-R is shown in SEQ ID NO:6.

[0639] Example 2 Design and synthesis of gRNA targeting vaccinia virus F1L region and Cas9 co-expression plasmid pX458del- 616gRNA

[0640] Design on the website http: / / crispor.tefor.net / , analyze the gene structure to select the appropriate target, and avoid the formation of shorter translation products. Enter the targeted F1L region sequence into the website, sort all the gRNA sequences listed in the website according to the "Doench'16-Score" parameter from small to large, and screen out the appropriate gRNA, whose sequence is shown in SEQ ID NO:8 and is named 616gRNA. Add BbsⅠ restriction sites to the 5' end of the forward direction and the 5' end of the reverse complementary sequence at both ends of the sequence, and send them to the company for synthesis. After phosphorylation modification and high-temperature denaturation, the synthesized sequence is gradually annealed to form a double-stranded DNA oligonucleotide. It and the gRNA and Cas9 co-expression vector plasmid pX458del were digested and recovered with BbsⅠ, reacted at 37℃ for 1.5h, and the gRNA sequence and linearized plasmid pX458del were obtained. After recovery by the kit, the two were connected to construct the plasmid pX458del-616gRNA.

[0641] After phosphorylation modification and high temperature denaturation, the double-stranded DNA oligonucleotide is gradually annealed to form a sequence as shown in SEQ ID NO: 12.

[0642] The sequence of plasmid pX458del-616gRNA is shown in SEQ ID NO:10.

[0643] The sequence of the Cas9 co-expression vector plasmid pX458del is shown in SEQ ID NO:9.

[0644] Example 3 Construction of recombinant virus NTV-ΔF1L-C7L

[0645] BHK-21 cells were inoculated in a 6-well cell culture plate. When the cells grew to 50% to 6%, the culture medium was replaced with DMEM medium containing 2% FBS. The plasmid pX458del-616gRNA was transfected with the transfection reagent X-tremeGENE HP DNA, and the cells were cultured at 37°C until the next day. When the BHK cells grew to 90%, vaccinia virus NTV was inoculated at 0.01 MOI. After 2 hours of viral infection, the culture supernatant was discarded and washed twice with PBS, and DMEM medium containing 2% FBS was added again. 2 μg of the recombinant plasmid pUC57-ΔF1L-LacZ-C7L was transfected in the same way, and the cells and supernatant were harvested 48 hours later and frozen at -70°C.

[0646] The sequence of the recombinant plasmid pUC57-ΔF1L-LacZ-C7L is shown in SEQ ID NO:7.

[0647] The nucleotide sequence of the recombinant region of the recombinant virus NTV-ΔF1L-C7L is shown in SEQ ID NO:11.

[0648] Example 4 Purification and Identification of Recombinant Virus NTV-ΔF1L-C7L

[0649] After the prepared NTV-ΔF1L-C7L recombinant solution was frozen and thawed three times, the single-spot solution was inoculated into BHK-21 cells grown to 90%, and the spots were spread with DMEM medium containing 1.3% agar, 50 μg / mL neutral red, 200 μg / mL X-gal and 2% FBS, and the blue single spots were picked and repeatedly inoculated into BHK-21 cells. Repeat 5 to 7 times until the white single spots no longer appear for two consecutive generations.

[0650] Take 20 μL of purified recombinant virus NTV-ΔF1L-C7L and use Qiagen blood / tissue genomic DNA extraction kit to extract viral DNA as a template for PCR identification. After the PCR reaction is completed, add 4 μL 6× Loading Buffer, mix well, and take 10 μL to observe the enzyme cleavage bands after agarose gel electrophoresis.

[0651] BHK-21 cells were infected with recombinant vaccinia virus NTV-ΔF1L-C7L at 5 MOI, and cells were harvested 12 hours after infection to prepare Western blot samples. After 15% SDS-PAGE gel electrophoresis, the separated proteins were electrotransferred to nitrocellulose (NC) membranes and blocked in 5% skim milk at room temperature for 2 hours. The primary antibody was F1 protein mouse antibody serum, and the secondary antibodies were Anti-Mouse IgG (H+L) DyLight 800 and Anti-rabbit IgG (H+L) DyLight 680, and then analyzed using the Odyssey infrared fluorescence scanning imaging system.

[0652] Example 5 Amplification of the recombinant virus NTV-ΔF1L-C7L

[0653] The virus solution was inoculated at 0.01 MOI into several bottles growing at 175 cm 2 Virus amplification was performed in BHK cells in the culture flask. When all cells were diseased (48-72 hours) and not detached, the cells were scraped off with a cell scraper, the culture medium was removed and centrifuged at 2500rpm for 10 minutes to collect the cell precipitate as much as possible, and the supernatant was discarded. The cell precipitate was washed once with 10mM Tris-HCl at pH 9.0, centrifuged and the supernatant was discarded, and 10mM Tris-HCl at pH 9.0 was added again. After freezing and thawing 3 times, the virus liquid was ultrasonically treated with a power of 60W, 4s each time, 8s interval, and 30 times in total. Then, trypsin was added at a ratio of virus liquid: 0.25% trypsin (without EDTA) = 4:1, and incubated in a constant temperature water bath at 37℃ for 30 minutes, with shaking once every 10 minutes. The digested virus liquid was centrifuged at 2500rpm for 10 minutes, the supernatant was transferred to a new centrifuge tube, the precipitate was washed 3 times with Tris-HCl and all the supernatants were mixed. Add 18mL of 36% sucrose solution to the ultracentrifuge tube, slowly drop the Tris-HCl containing the virus onto the upper layer of the sucrose solution, and separate the Tris-HCl solution and the sucrose solution. Use Tris-HCl to adjust the ultracentrifuge tube to the same mass, and place it in an ultracentrifuge for ultracentrifugation at 13500rpm for 90min. Carefully discard the supernatant, resuspend the precipitate with PBS, take a portion of the virus and determine the virus titer by plating, and transfer the remaining virus to a cryopreservation tube and freeze it at -70℃ for later use.

[0654] Example 6 Animal toxicity test of recombinant virus NTV-ΔF1L-C7L

[0655] The virulence of recombinant vaccinia virus NTV-ΔF1L-C7L in BALB / c mice was studied by nasal inoculation to simulate upper respiratory tract infection. Female BALB / c mice aged 6-8 weeks were randomly divided into groups and infected with NTV (10 6 , 10 7 , 5×10 7 PFU / 40μL), NTV-ΔF1L-C7L (10 6 , 10 7 , 5×10 7 PFU / 40μL) and VTT (10 6 , 10 7PFU / 40μL), and the control group was inoculated with an equal volume of PBS (40μL). The weight of the mice was recorded and the changes in the physical signs of the mice were observed for 14 consecutive days. When the intranasal inoculation dose was 106PFU, the weight loss in the VTT group reached the maximum on the 7th day, and on the 8th day, 3 mice lost more than 30% of their weight and were euthanized. The weight loss in the NTV group reached the maximum on the 1st day, reaching 6.14%, while there was no significant decrease in weight in the NTV-ΔF1L-C7L group, and there was a significant statistical difference between the two (P<0.01); when the intranasal inoculation dose was 107PFU, the weight loss in the VTT group reached the maximum on the 6th day, and on the 7th day, 4 mice lost more than 30% of their weight and all died on the 8th day. The weight loss of the NTV group reached the maximum on the second day, reaching 3.59%, and the weight loss of NTV-ΔF1L-C7L 107 reached the maximum on the third day, reaching 1.55%, and there was no statistical difference between the two (P>0.05); when the intranasal inoculation dose was 5×107PFU. The weight loss of the NTV group reached the maximum on the third day, reaching 14.55%, and the weight loss of the NTV-ΔF1L-C7L group reached the maximum on the fourth day, reaching 15.16%, and there was no statistical difference between the two (P>0.05). This shows that the recombinant virus NTV-ΔF1L-C7L has similar virulence to the original virus NTV.

[0656] Example 7 Animal Immunization Experiment of Recombinant Virus NTV-ΔF1L-C7L

[0657] Female BALB / c mice aged 6-8 weeks were randomly divided into groups and infected with NTV (10 7 PFU / 100μL), NTV-ΔF1L-C7L (10 7 PFU / 100μL) and VTT (10 6 PFU / 100μL), and the control group was inoculated with an equal volume of PBS (100μL). After the initial immunization on day 0 and the booster immunization on day 14, the mouse serum was collected under sterile conditions on day 28 and the spleen was dissected to prepare a lymphocyte suspension. The CD8+T cell epitope vaccinia virus E3 (VGPSNSPTF) and F2 (SPYAAGYDL) peptides in BALB / c mice were used as stimuli, and the changes in IFN-γ levels were detected by ELISPOT detection kit. After stimulation with E3 peptide, the IFN-γ level induced by NTV-ΔF1L-C7L group was the highest, which was about 3 times that of the IFN-γ level induced by NTV group, and the difference was statistically significant (P<0.05); while after stimulation with F2 peptide, the IFN-γ level induced by VTT group was the highest, followed by NTV-ΔF1L-C7L group, and the difference was also statistically significant compared with NTV group (P<0.05).

[0658] After the vaccinia virus antigen was coated on a 96-well plate, the ELISA method was used to detect the specific antibody IgG titer of each group of mice. The antibody titer of the recombinant vaccinia virus NTV-ΔF1L-C7L group could reach 1.9×10 4 , while the antibody titers induced by NTV and VTT were 7.3×10 3 and 9.7×10 3 The difference was statistically significant compared with the NTV-ΔF1L-C7L group (P<0.05). The ELISA method was also used to detect the antibody IgG1 and IgG2a titers of mice in each group. All viruses induced higher IgG2a antibody titers, which means that the immunity induced by vaccinia virus is more inclined to the Th1 cell immune response that secretes IFN-γ and IL-2.

[0659] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A method for preparing a CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain, characterized in that: The preparation method comprises the following steps: (1) Construction of recombinant plasmid of vaccinia virus; (2) Construction of a CRISPR / Cas9 system targeting vaccinia virus; (3) Construction of recombinant vaccinia virus; (4) purifying and identifying vaccinia recombinant viruses; and (5) Amplifying the vaccinia recombinant virus to obtain the CRISPR / Cas9-based vaccinia virus gene-modified vaccine strain.

2. The preparation method according to claim 1, characterized in that: The step (1) further includes: (A) preparing a first vector plasmid; (B) digesting the first vector plasmid and the vaccinia virus TK region recombinant plasmid of step (A) respectively to obtain the selection marker LacZ gene and the first linearized plasmid, recovering and connecting the two to construct a second vector plasmid; and (C) using primers C7L-F and C7L-R, and the replicative vaccinia virus Tiantan strain as a template, obtaining the PCR product C7L gene and connecting it to a vector to obtain a third vector plasmid, digesting the second vector plasmid and the third vector plasmid obtained in step (B) respectively to obtain the C7L gene and the second linearized plasmid, and connecting the two after recovery to construct the recombinant plasmid of the vaccinia virus; Preferably, in the step (B), the vaccinia virus TK region recombinant plasmid is a vaccinia virus Tiantan strain TK region recombinant plasmid; Preferably, the step (A) further comprises: selecting the upstream sequence of the F1L gene as the recombinant left arm and the downstream sequence of the F1L gene as the recombinant right arm according to the vaccinia virus NTV genome sequence, synthesizing the sequence and inserting it into a plasmid, thereby obtaining the first vector plasmid; Preferably, the vaccinia virus early and late promoters are contained between the recombinant left arm and the recombinant right arm, and the sequence of the vaccinia virus early and late promoters is more preferably SEQ ID NO: 4 and / or SEQ ID NO: 13; Preferably, the upstream sequence of the F1L gene is SEQ ID NO: 1 and / or SEQ ID NO: 3; Preferably, the downstream sequence of the F1L gene is SEQ ID NO: 2; and / or Preferably, the plasmid is pUC57, and its sequence is SEQ ID NO:

14.

3. The preparation method according to claim 1 or 2, characterized in that: In the step (C), before digestion, the steps include: the two ends of the PCR contain restriction sites, and the second vector plasmid and the third vector plasmid obtained in the step (B) are respectively digested by water bathing with the restriction sites; Preferably, the restriction site is BamHI; Preferably, the temperature of the water bath is 25-50°C, more preferably 30-40°C, most preferably 37°C; Preferably, the digestion time is 0.5 to 3 hours, more preferably 0.5 to 2 hours, most preferably 1 hour; Preferably, in the step (C), the vector is pMD18-T; Preferably, in step (C), the sequence of the primer C7L-F is SEQ ID NO: 5, and the sequence of the primer C7L-R is SEQ ID NO: 6; and / or Preferably, in the step (C), the nucleotide sequence of the recombinant plasmid of the vaccinia virus is as shown in SEQ ID NO:

7.

4. The preparation method according to any one of claims 1 to 3, characterized in that In the step (2), the CRISPR / Cas9 system targeting vaccinia virus comprises: a plasmid for co-expression of gRNA targeting vaccinia virus and Cas9 gene; Preferably, the plasmid for co-expression of the gRNA and Cas9 gene targeting vaccinia virus includes a plasmid for co-expression of the gRNA and Cas9 gene having the sequence shown in SEQ ID NO:

10.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In the step (2), the method for preparing the CRISPR / Cas9 system targeting vaccinia virus comprises: (D) Screening of gRNA expression plasmids targeting vaccinia virus; (E) synthesizing the gRNA expression plasmid sequence screened in step (D), and forming a DNA oligonucleotide after phosphorylation modification and high temperature denaturation; and (F) digesting and recovering the DNA oligonucleotide prepared in step (E) and the gRNA expression plasmid and Cas9 gene expression vector plasmid screened in step (D) to obtain the gRNA sequence and the linearized Cas9 gene expression vector plasmid, and connecting the two after recovery to construct the CRISPR / Cas9 system targeting vaccinia virus; Preferably, in the step (D), the gRNA targeting vaccinia virus comprises an expression plasmid expressing a gRNA having a sequence shown in SEQ ID NO: 8; Preferably, in the step (E), the DNA oligonucleotide comprises a DNA oligonucleotide expressing a sequence shown in SEQ ID NO: 12; Preferably, in the step (F), the nucleotide sequence of the Cas9 gene expression vector plasmid is as shown in SEQ ID NO:

9.

6. The preparation method according to any one of claims 1 to 5, characterized in that In step (3), the operation of constructing the vaccinia recombinant virus further comprises the following steps: (a) growing cells; (b) transfecting the CRISPR / Cas9 system targeting vaccinia virus using a transfection reagent and inoculating non-replicating vaccinia virus; and (c) transfecting a recombinant plasmid of vaccinia virus and inoculating a non-replicating vaccinia virus, harvesting cells and supernatant, and freezing to obtain the recombinant vaccinia virus; Preferably, the cell in step (a) is BHK-21; Preferably, in step (b), the transfection reagent is X-tremeGENE HP DNA; Preferably, in step (c), the nucleotide sequence of the recombinant region of the vaccinia recombinant virus is as shown in SEQ ID NO: 11; and / or Preferably, in step (b) and step (c), the non-replicating vaccinia virus is NTV.

7. The preparation method according to any one of claims 1 to 6, characterized in that The step (4) further comprises: thawing the recombinant vaccinia virus and inoculating it into BHK-21 cells for purification and identification; Preferably, the number of thawing is 2 to 5 times, more preferably 2 to 4 times, and most preferably 3 times; Preferably, the BHK-21 cells are BHK-21 cells grown to 80-95%, more preferably BHK-21 cells grown to 85-95%, and most preferably BHK-21 cells grown to 90%; and / or Preferably, the identification includes: observing enzyme cleavage bands and analyzing with an infrared fluorescence scanning imaging system.

8. The preparation method according to any one of claims 1 to 7, characterized in that The step (5) further comprises the following steps: (d) inoculating the vaccinia recombinant virus into cells, centrifuging after culturing, discarding the supernatant, collecting the cell precipitate, washing and centrifuging the cell precipitate, and sonicating after thawing; (e) adding pancreatin to the sonicated vaccinia recombinant virus solution, incubating and digesting, and then centrifuging; the pancreatin is preferably EDTA-free pancreatin; (f) adding sucrose solution to separate the layers, centrifuging, discarding the supernatant, resuspending the precipitate, and storing it for later use; Preferably, in step (d), the ultrasonic power is 50-70W, the time is 3-5s, the interval is 6-9s, and the number is 25-35 times, and the most preferred ultrasonic power is 60W, the time is 4s, the interval is 8s, and the number is 30 times; and / or Preferably, in step (f), the storage temperature is -90 to -50°C, more preferably -80 to -60°C, most preferably -70°C.

9. A genetically modified vaccine strain of vaccinia virus, characterized in that: The vaccinia virus gene-modified vaccine strain is prepared by the preparation method described in any one of claims 1 to 8.

10. Use of the vaccinia virus gene-modified vaccine strain according to claim 9 in the preparation of monkeypox vaccine products or smallpox vaccine replacement products.

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