CN113559134B

CN113559134B - Medicine for treating tumor

Info

Publication number: CN113559134B
Application number: CN202110867976.9A
Authority: CN
Inventors: 关继羽; 赵魁; 贺文琦; 林静; 孙诗惠; 高飞
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2023-11-03
Anticipated expiration: 2041-07-29
Also published as: CN113559134A

Abstract

The application provides a medicine for treating tumors, which comprises an attenuated strain of sheep infectious impetigo virus. The virulence gene deletion strain of the infectious impetigo virus of sheep is prepared by deletion of virulence genes of ORFV001-ORFV008, ORFV020, ORFV024, ORFV073, ORFV112-ORFV131 and ORFV134, namely the attenuated strain of the infectious impetigo virus of sheep. The attenuated strain of the sheep infectious impetigo virus is used for anti-tumor drugs, preventing tumor cell growth and promoting tumor cell death; preventing in situ tumor growth; preventing tumor metastasis; increasing the immune cell proportion in the tumor.

Description

Medicine for treating tumor

Technical Field

The application relates to a new application of an attenuated strain of an infectious impetigo virus, in particular to an application of an attenuated strain of an infectious impetigo virus caused by gene deletion in resisting tumors.

Background

The neoplastic disease is not only the second biggest killer for human health, but also the first ten causes of death in companion animals such as dogs and cats, and has significant harm to both human and animal health. Furthermore, immunotherapy and chemotherapy treatment against neoplastic diseases add a heavy economic burden to the life of people. At present, personalized treatment schemes for specific tumors are endless. Radiation and chemotherapy regimens are broad spectrum effective, but have significant side effects; various small molecule inhibitor therapies are targeted, but are costly and prone to developing drug resistance; immunotherapy such as PD1 antibody and PDL1 antibody is effective, but has little adaptation; the effect of CAR-T therapy is significant, but the incidence of side effects is high. Oncolytic viruses are a new type of antitumor therapy which has been paid attention in recent years, and by genetically modifying viruses, the virus virulence is weakened, and meanwhile, the killing capacity to tumor cells and the capacity of changing tumor immunity microenvironment can be maintained.

Infectious impetigo virus (ORFV) belongs to the family poxviridae, members of the parapoxviridae family. The research shows that ORFV has strong immunoregulation capability, and can reduce hepatic fibrosis injury and pathogenicity of certain viruses. Furthermore, ORFV has also been demonstrated to alter tumor immune microenvironment, promoting secretion of cytokines, recruiting NK cells, dendritic cells and phagocytes. At the same time, researchers have also tried to deliver immunogenic genes as vectors with ORFV and construct recombinant vaccines. Therefore, the method for optimizing the immunoregulation capacity and the targeted gene transfer capacity of the ORFV by constructing the genetically modified strain has wide application prospect. Amplification and purification of ORFV is an important step in subsequent application-based research. The virus has been successfully amplified in MDBK (bovine kidney cells), OFTu cells (sheep embryo turbinates) and Vero (African green monkey kidney cells), concentrated by ultracentrifugation, followed by sucrose density gradient method for virus purification, and finally identification of virus particles by transmission electron microscopy.

Disclosure of Invention

The application provides a medicine for treating tumors, which comprises an attenuated strain of sheep infectious impetigo virus. The attenuated strain of the sheep infectious impetigo virus is used as an immunostimulant or enhancer to stimulate an innate immune response of an organism and stimulate the organism to generate an anti-tumor immune response.

Preferably, the attenuated strain of the infectious impetigo sheep virus (also called attenuated strain of ORFV with virulence gene deletion) is prepared by virulence gene deletion of the infectious impetigo sheep virus. The ORFV virulence gene-deficient strain can be used as an immunostimulant or enhancer to stimulate an innate immune response of an organism. The ORFV virulence gene-deleted attenuated strain can be used for treating (inhibiting) aspects of tumor growth and metastasis.

According to the test results, after the ORFV virulence gene-deficiency attenuated strain is treated by abdominal cavity (i.p.), tail vein (i.v.) and intramuscular injection (i.m.), the tumor lung metastasis model of the mice can obviously reduce the tumor focus in the lung, simultaneously reduce the clinical symptoms of the mice and reduce the death rate. The ORFV virulence gene-deleted attenuated strain significantly reduced tumor foci size following intratumoral (i.t.), intraperitoneal (i.p.), caudal vein (i.v.), and intramuscular (i.m.) treatment of mice subcutaneous tumor models. The result shows that the strain with the attenuated ORFV virulence gene can induce the body to generate an innate immune response, thereby playing an anti-tumor role. Based on the above test results, the inventors propose to treat neoplastic diseases by injection of attenuated strains of ORFV virulence gene deletion.

Any of the above is preferred to be a strain obtained by deleting any virulence gene at both ends or in the middle of the ORFV genome in the case of causing a virulence deletion of the ORFV.

Preferably, any of the above, the virulence genes comprise at least one of ORFV001-ORFV008, ORFV020, ORFV024, ORFV073, ORFV112-ORFV 134. The virulence gene of the application refers to a gene related to the generation of virulence factors (from the second edition of microbiology noun).

Any of the above is preferably as set forth in SEQ NO:20 comprising virulence genes ORFV001, ORFV005, ORFV007, ORFV008; as shown in SEQ NO:21, comprising virulence genes ORFV112, ORFV113, ORFV114, ORFV115, ORFV116, ORFV117, ORFV118, ORFV119, ORFV120, ORFV121, ORFV122, ORFV123, ORFV124, ORFV125, ORFV126, ORFV127, ORFV128, ORFV129, ORFV130, ORFV131, ORFV132, ORFV134; the method for preparing the virulence gene deletion of the sheep infectious impetigo virus comprises the virulence gene deletion method as set forth in any one of (1) to (7) below:

(1) As shown in SEQ NO:20, a deletion of the nucleotide sequences of virulence genes ORFV N1 through ORFV N2, including a deletion of the nucleotide sequences of virulence genes and a deletion of the nucleotide sequences of gaps between virulence genes, wherein N1, N2 are virulence gene numbers, n1=001, 005, 007, n2=005, 007, 008, N2> N1;

(2) As shown in SEQ NO:20, or a deletion of only the virulence gene without the inclusion of the spacer nucleotide sequence; preferably, the deletion fragment is a incompletely contiguous deletion: the deletion segment is at least two sequences, wherein the deletion segment comprises continuous segments containing nucleotide sequences of virulence genes and interval sequences between virulence genes and deletion of virulence gene segments discontinuous with other deleted virulence genes; preferred deletion fragments are completely discontinuous deletions: any virulence genes deleted are discontinuous.

(3) As shown in SEQ NO:21, a deletion of the nucleotide sequences of virulence genes ORFV M1 through ORFV M2, including a deletion of the nucleotide sequences of the virulence genes and a deletion of the nucleotide sequences of the gaps between the virulence genes, wherein M1, M2 are virulence gene numbers, m1=112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, m2=113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 134, M2> M1; further M1, M2 are preferably: m1=112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, m2=113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 134, M2> M1.

(4) As shown in SEQ NO:21, a co-deletion of at least any 2 virulence genes in the nucleotide sequence set forth in seq id no; the co-deletion of at least any 2 virulence genes may be a deletion of the nucleotide sequences of the virulence genes and a deletion of the nucleotide sequences of the gaps between the virulence genes, or may be a deletion of only the virulence genes without the inclusion of the nucleotide sequences of the gaps; preferably, the deletion fragment is a incompletely contiguous deletion: the deletion segment is at least two sequences, wherein the deletion segment comprises continuous segments containing nucleotide sequences of virulence genes and interval sequences between virulence genes and deletion of virulence gene segments discontinuous with other deleted virulence genes; preferred deletion fragments are completely discontinuous deletions: a discontinuity between any virulence genes deleted;

(5) Setting SEQ NO:20 and the nucleotide sequence shown in SEQ NO:21, and simultaneously carrying out deletion mutation on the nucleotide sequence containing at least one virulence gene in the nucleotide sequence shown in the formula (1);

(6) Setting SEQ NO: 22. SEQ NO: 23. SEQ NO:24, and at least one of the virulence gene nucleotide sequences set forth in SEQ NO: 20. SEQ NO:21, and simultaneously performing deletion mutation on at least one of the nucleotide sequences of the virulence genes shown in figure 21.

(7) Setting SEQ NO: 22. SEQ NO: 23. SEQ NO:24, and simultaneously performing deletion mutations on at least two of the virulence gene nucleotide sequences shown in figure 24.

Preferably, any of the above has the amino acid sequence of ORFV020 as set forth in SEQ NO:22, the nucleotide sequence is shown as SEQ NO: shown at 25; the amino acid sequence of ORFV024 is shown in SEQ NO:23, the nucleotide sequence is shown as SEQ NO: 26; the amino acid sequence of ORFV073 is shown in SEQ NO:24, the nucleotide sequence is shown as SEQ NO: shown at 27. The mutation method is carried out by conventional means in the art, and is not described in detail herein.

Preferably, any one of the above-mentioned attenuated strains of the sheep infectious impetigo virus is an ORFV120 gene-deleted strain in which the virulence gene ORFV120 is deleted.

Preferably, any of the above deletion mutations do not include a mutation of the ORFV132 gene.

Preferably, any one of the above deletion mutations is not a deletion mutation of a single gene of the ORFV132 gene.

Preferably, any one of the above is that the attenuated strain of the sheep infectious impetigo virus is a lyophilized formulation.

Any of the above is preferred to include 0-10 parts by weight of the attenuated strain of the infectious impetigo virus.

Preferably, any of the above is a suspension formulation comprising the attenuated strain of sheep infectious impetigo virus and a stabilizer. The stabilizer includes, but is not limited to, at least one of the following: various sugars such as lactose, glycerol, sucrose, mannitol, trehalose, fructose, galactose, glucose, etc.; various amino acids; dextran, polyethylene glycol, and the like.

Preferably, the mass ratio of the attenuated strain of the sheep infectious impetigo virus to the stabilizer is 1: 1-1: 100. it is further preferred that the mass ratio of the attenuated strain of the sheep infectious impetigo virus to the stabilizer is 1:1,1:10,1:20,1:30,1:40,1:50,1:60,1:70,1:80,1:90,1:100.

in any of the above, it is preferable that the attenuated strain of ORFV virulence gene is used as a main component. Further preferably, the strain comprises 50. Mu.g of ORFV virulence gene-attenuated strain and 50. Mu.g of stabilizer.

Preferably, any of the above drugs is mixed with an injectable carrier to prepare an injectable preparation. The carrier for injection is preferably physiological saline. When in use, physiological saline is added into the medicine to prepare injection preparation.

Preferably, any of the above is administered by at least one of intratumoral injection, intramuscular injection, intraperitoneal injection or intravenous injection.

Preferably, any of the above drugs inhibit in situ growth of a tumor and/or inhibit metastasis of a tumor.

Preferably, any of the above is administered at a dose of 10 per individual ³ -10 ⁹ TCID ₅₀ More preferably 10 ³ -10 ⁷ TCID ₅₀ Preferably 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ TCID ₅₀ 。

Preferably, any of the above drugs is used in combination with at least one of anti-tumor radiotherapy, anti-tumor chemotherapy, anti-tumor targeting drugs, anti-tumor hormone therapy and anti-tumor immunotherapy.

Preferably, any of the above drugs according to the application have the following antitumor activity:

(1) Preventing the growth of tumor cells and promoting the death of the tumor cells in an in vitro test;

(2) Preventing in situ tumor growth;

(3) Preventing tumor metastasis;

(4) Increasing the proportion of immune cells within a tumor, in a preferred embodiment of the application, demonstrates that the proportion of T lymphocytes within a tumor can be significantly increased;

(5) Can be used in combination with chemotherapeutic agents;

(6) Can be used in combination with radiotherapy;

(7) The compound can be combined with anti-tumor immune medicines such as PD-1 antibody, PDL-1 antibody and the like, and the preferable combined use mode comprises the steps of administering the PD-1 antibody and the PDL-1 antibody by intraperitoneal injection, wherein the preferable dosage is 2.5-10mg/kg according to different tumors; the effect of the missing toxin therein is to recruit immune cells.

Preferably, any one of the above-mentioned ORFV is an ORFV-SY17 strain, an ORFV-NA17 strain or an ORFV-Jilin strain.

The preferred wild strains ORFV-SY17, ORFV-NA17 and ORFV-Jilin strains of the application are strains disclosed in the prior art and are publicly available in a shared manner with the authors. ( Zhong J#, guan J#, zhou Y, cui S, wang Z, zhou S, xu M, wei X, gao Y, zhai S, song D, he W, gao F, zhao K. Genomic characterization of two Orf virus isolates from Jilin province in China. Viruses Genes,2019,55:490-501.2.Zhao K,Song D,He W,Lu H,Zhang B,Li C,Chen K,Gao F.Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in sheep in the Jilin province of China.Vet Microbiol.2010May19;142 (3-4):408-15. )

Preferably, any one of the above is used in combination with conventional tumor therapeutic drugs for improving the immunity of the body, so that a better prognosis is obtained after treatment.

The medicine has obvious effect of treating tumor: (1) Inhibiting in-situ growth of tumor by intratumoral injection and intramuscular injection; (2) Tumor metastasis is inhibited by intraperitoneal injection and intramuscular injection.

Drawings

FIG. 1 is a transmission electron microscope examination of an ORFV120 gene-deleted strain in example 1 of the present application.

FIG. 2 shows the change in body weight of mice with the ORFV120 gene-deleted strain of example 1 of the present application before and after intramuscular injection.

FIG. 3 shows the changes in immunocytes before and after intratumoral injection of ORFV120 gene-deleted strain of example 1 of the present application.

FIG. 4 shows the weight and volume changes of tumor cells of the ORFV120 gene-deleted intratumoral injection form of example 1 of the present application after tumor-bearing mice are treated.

FIG. 5 shows the change in the number of metastases in the lung after treatment of mice bearing the metastases in the form of intravenous injection of ORFV120 gene-deleted strain of example 1 according to the application.

Detailed Description

The present application will be more clearly and fully described by the following examples, which are intended to be illustrative of only some, but not all, of the examples. The examples are presented to aid in understanding the application and should not be construed to limit the scope of the application in any way.

Example 1

Preparation of ORFV120 gene-deleted strain:

the ORFV120 gene-deleted strain of the application is prepared as follows:

(1) Preparing a parent strain: the parent strain is an infectious impetigo virus ORFV-SY17 strain, and the full-length gene sequence of the infectious impetigo virus ORFV-SY17 strain is as GenBank: MG 712417.1;

(2) Construction of the screening expression cassette: inserting a specific promoter and a first marker gene applicable to the sheep infectious impetigo virus into a vector framework to obtain a screening expression cassette;

(3) Construction of shuttle plasmid: amplifying sequences at two sides of an ORFV120 gene deletion sequence of the ORFV-SY17 strain to serve as recombinant homology arms, cloning the sequences into the screening expression cassette constructed in the step (2), and obtaining a marked recombinant shuttle plasmid;

(4) Transfecting a host cell infected with the ORFV-SY17 wild strain with the shuttle plasmid prepared in the step (3) to obtain a deleted virus expressing the first marker gene;

(5) Infecting a host cell with the deletion virus expressing the first marker gene prepared in the step (4), and transfecting the host cell infected with the deletion virus expressing the first marker gene with a plasmid containing only the recombinant homology arm of the ORFV120 gene to obtain a deletion strain without the first marker gene.

Wherein, preferably, the ORFV120 gene comprises the amino acid sequence of SEQ NO:1, the mutation mode of the ORF120 gene comprises the nucleotide sequence shown in SEQ NO:1 from nucleotide sequence 1 to nucleotide sequence 86 to nucleotide sequence 476 to nucleotide sequence 588. Preferred modes of mutation include: SEQ NO:1, 1 to 476 nucleotides, 1 to 477 nucleotides, 1 to 478 nucleotides, … …,1 to 588 nucleotides in the nucleotide sequence shown in 1; nucleotides 2 to 476, nucleotides 2 to 477, nucleotides 2 to 478, … …, nucleotides 2 to 588; nucleotides 3 to 476, nucleotides 3 to 477, nucleotides 3 to 478, … …, nucleotides 3 to 588; nucleotides 4 to 476, nucleotides 4 to 477, nucleotides 4 to 478, … …, nucleotides 4 to 588; such deletion mutations up to nucleotides 86 to 476, nucleotides 86 to 477, nucleotides 86 to 478, … …, nucleotides 86 to 588 are deduced from the sequence of SEQ NO:1, wherein the nucleotide sequence between the N1 base and the N2 base in the nucleotide sequence is knocked out, N1 is more than or equal to 1 and less than or equal to 86, N2 is more than or equal to 476 and less than or equal to 588; another preferred variant is to divide the sequence of SEQ NO:1 to the N2 base in the nucleotide sequence shown in the above-mentioned reference numeral, and other nucleotide fragments are substituted for the nucleotide sequence between the N1 base and the N2 base, and in a preferred embodiment of the present application, the deletion fragment is substituted for the nucleotide sequence of the marker gene. It is further preferred that the mutated form of the ORFV120 gene further comprises the amino acid sequence of SEQ NO:1 to the 86 th to 476 th nucleotides in the nucleotide sequence shown in the formula.

In this example, the deletion mutation construction mode is finally selected, i.e. the start site of the determined knockout sequence (i.e. the deletion sequence) is located in SEQ NO:1, and a termination site is positioned between the 1 st base and the 86 th base of the nucleotide sequence shown in the formula 1, and between the 476 th base and the 588 th base of the nucleotide sequence, wherein the most preferred knockout sequence is SEQ NO:1, namely the 86 th to 476 th bases of the nucleotide sequence shown in the formula 1, namely the 86 th base is the initial base of the knocked-out sequence, the 476 th base is the termination base, namely the nucleotide sequence shown in the SEQ NO:2, and a nucleotide sequence shown in the following formula. Further preferred is to divide the sequence of SEQ NO:2, and inserting the nucleotide sequence of the marker gene after knocking out the nucleotide sequence shown in the figure. In a final preferred embodiment, the nucleotide sequence of the inserted marker gene is further removed by recombination, and the method of removing the nucleotide sequence of the inserted marker gene by recombination is a method conventional in the art, and will not be described in detail herein.

The preparation method of the preparation method reference 2020115283418 in this embodiment specifically comprises the following steps:

examples sources of materials:

cells and viruses: original sourceThe turbinate bone cells (OFTu) of the substituted foetus sheep are separated from healthy pregnant sheep of 3-5 months of gestation, the pregnant sheep are anesthetized, the foetus sheep are aseptically taken out, the turbinate bone tissue is collected and sheared, the turbinate bone tissue is washed for 2-3 times by using D-Hanks liquid, 0.25% trypsin is added for digestion treatment for 30min, and then the digestion is repeated for 1-2 times. Collecting cell suspension, centrifuging at low speed, discarding supernatant, re-suspending cells in MEM medium containing 10% FBS, and standing at 37deg.C in 5% CO ₂ Culturing in an incubator. OFTu cells were used for plasmid transfection and virus recombination experiments. ORFV-SY17 strain is a Jilin university veterinary pathology laboratory save strain, disclosed in Zhong J#, guan J#, zhou Y, cui S, wang Z, zhou S, xu M, wei X, gao Y, zhai S, song D, he W, gao F, zhao K. Genomic characterization of two Orf virus isolates from Jilin province in China. Virus Genes,2019,55:490-501, a publicly available virus source with a viral titer of 107.37TCID ₅₀ Packaging and storing at-80deg.C.

1.1 construction of EGFP Screen expression cassettes

First, a VV p7.5 promoter sequence fragment was amplified by a template PCR method using pSPV-EGFP plasmid (supplied by the university of south medical science Biotechnology laboratory). Designing a specific amplification primer aiming at the promoter fragment (the nucleotide sequence of an upstream amplification primer VV p7.5 Fw is shown as SEQ NO:3, the nucleotide sequence of a downstream amplification primer VV p7.5 Rv is shown as SEQ NO: 4), and PCR (polymerase chain reaction) amplification conditions are 98 ℃ 30s,55 ℃ 15s,72 ℃ 5s and 34 cycles; next, EGFP sequence fragments were amplified using pEGFP-N1 vector as a template. Specific amplification primers for EGFP (the nucleotide sequence of an upstream primer EGFP Fw is shown as SEQ NO:5, and the nucleotide sequence of a downstream primer EGFP Rv is shown as SEQ NO: 6) are designed, and PCR amplification conditions are 98 ℃ 30s,55 ℃ 15s,72 ℃ 8s and 34 cycles. Connecting the VV p7.5 promoter sequence fragment obtained by PCR amplification and the EGFP sequence amplification fragment by a fusion PCR method, wherein the upstream primer is a nucleotide sequence of VV p7.5 Fw as shown in SEQ NO:3, the nucleotide sequence of the downstream primer EGFP Rv is shown as SEQ NO:6, the PCR fusion amplification conditions are 98 ℃ for 30s,55 ℃ for 15s,72 ℃ for 60s and 34 cycles; the recovered and purified VV p7.5-EGFP fusion gene fragment is inserted into pUC57 skeleton plasmid, and the correctness of the recombinant vector sequence is verified by sequencing, so that the pUC57-EGFP screening expression cassette is obtained.

1.2 construction of recombinant shuttle plasmid for ORFV120 Gene deletion

And determining left and right homology arm sequences according to the upstream and downstream sequence information of the ORFV120 gene, and designing primers according to the homology arm sequences. The left homology arm (LF; 819 bp) and the right homology arm (RF; 852 bp) were amplified respectively (primer sequence: ORFV120-LF-Fw nucleotide sequence: SEQ NO: 7; ORFV120-LF-Rv nucleotide sequence: SEQ NO: 8; primer sequence: ORFV120-RF-Fw nucleotide sequence: SEQ NO: 9; ORFV120-RF-Rv nucleotide sequence: SEQ NO: 10). The PCR amplification conditions were 98℃30s,55℃15s,72℃10s,34 cycles. And cloning PCR amplified products of the left and right homology arms into pUC57-EGFP screening expression cassettes respectively, and verifying the recombinant shuttle plasmid through sequencing to obtain pUC57-LF delta 120-eGFP-RF delta 120 recombinant transfer plasmid. The deleted ORFV120 gene is a nucleotide sequence of 120779 to 121169 of the whole gene of the infectious impetigo virus of sheep, and the nucleotide sequence of the DNA sequence is shown in SEQ ID NO: 2.

1.3 screening and purification of ORFV120 Gene-deleted Virus

OFTu cells (cell number of about 2X 10) ⁵ And then the cells are spread in a 6-well plate, when the cell growth condition is good (about 70-80% confluence), the original strain of ORFV-SY17 (MOI=1-5) is inoculated without serum, after incubation for 1h at 37 ℃, DMEM complete culture solution containing 2-5% fetal bovine serum is added, and further culture is carried out for 2-3h. Then, 1-2. Mu.g of the purified pUC 57-LF.DELTA.120-eGFP-RF.DELTA.120 plasmid was mixed with 3-5. Mu.l of Lipofectamine ^TM After 3000 reagents are mixed, OFTu cells are transfected for 48 hours, and green fluorescence of the cells is generated. The appearance, duration and stable amplification of the green fluorescent signal indicate successful acquisition of the recombinant virus. The cells carrying the recombinant virus (carrying green fluorescence) were repeatedly freeze-thawed 3 times to release the recombinant virus, which was then inoculated into 96-well plates with OFTu cells for further screening. The cells with green fluorescence were subjected to dilution screening for 4-6 rounds to obtain purified recombinant virus, designated ORFV/Δ120/EGFP+. The 120779 to 121169 nucleotide is deleted relative to the full length sequence of the ORFV-SY17 strain of the infectious impetigo virus.

1.4PCR method for identifying ORFV/delta 120/EGFP+ recombinant virus

The method is used for verifying whether the virus ORFV120 gene is deleted successfully or not and whether the wild strain is polluted or not by using a specific primer for the virus ORFV120 gene (the nucleotide sequence of ORFV120-Fw is shown as SEQ NO:11, the nucleotide sequence of ORFV ORFV120-Rv is shown as SEQ NO: 12) and a specific primer for EGFP (the nucleotide sequence of EGFP-Fw is shown as SEQ NO:13 and the nucleotide sequence of EGFP-Rv is shown as SEQ NO: 14) to identify the purity of the wild ORFV and the ORFV/delta 120/EGFP+ virus genome DNA respectively by utilizing a virus genome extraction kit. The identification result shows that the amplification result of the ORFV120 gene of the recombinant virus is negative, and the amplification result of the EGFP gene is positive, indicating that the recombinant virus lacking the ORFV120 gene has been obtained and purified.

1.5 screening and purification of deleted strains deleted from the selectable marker Gene EGFP

Constructing a recombinant plasmid only comprising an ORFV120 gene recombinant homology arm, transfecting the recombinant plasmid into OFTu cells of the ORFV-SY17Δ120 deletion strain, selecting and purifying single non-fluorescent clone by the method, and performing multiple rounds of screening by the limiting dilution method to obtain the deletion ORFV120 strain without the first marker gene.

Description: example 1 the method of constructing an ORFV120 gene-deleted strain in the 2020115283418 patent application cited in this example is a prior study in the laboratory, and the related patent application is in the process of patent examination, and the method of constructing an ORFV120 gene-deleted strain is not the application itself. This patent is a further study of the prior patent application, and the focus of the protection of the present application is the application of the ORFV120 gene-deleted strain and other attenuated strains of infectious impetigo virus as antitumor agents.

The ORFV120 gene-deleted strain obtained in example 1 can be used alone as an antitumor drug.

Example 2 detection of anti-tumor Effect of ORFV120 Gene-deleted Strain

In example 2, the antitumor effect of the ORFV120 gene-deleted strain obtained in example 1 was examined.

1. Transmission electron microscope observation identification of ORFV120 gene deletion strain

And (5) placing the ORFV120 gene-deleted strain under a transmission electron microscope for observation and identification. The results are shown in FIG. 1.

2. Toxicity safety evaluation of ORFV120 Gene-deleted Strain

In the implementation process of the application, the ORFV120 gene-deleted strain is intramuscular injected into mice with the injection dosage of 2 multiplied by 10 ⁶ TCID ₅₀ The change in body weight of mice at day 0-15 of injection was recorded. The results are shown in figure 2, where the above viruses did not significantly alter the body weight of the mice.

3. Effect of ORFV120 Gene-deleted Strain therapeutic injection on B16 intratumoral immune cell recruitment

The application utilizes the intratumoral injection mouse tumor-bearing model of ORFV120 gene deletion strain, and observation is carried out after 3 intratumoral injection, and the injection dosage is 1 multiplied by 10 each time ⁶ TCID ₅₀ . The results are shown in FIG. 3, which can significantly increase the proportion of T lymphocytes in tumors. CD3 is shown in FIG. 3 ⁺ T lymphocyte increase and CD8 ⁺ T lymphocytes increase.

4. Effect of ORFV120 gene-deleted strain therapeutic injection on B16 tumor growth

The application utilizes the intratumoral injection mouse tumor-bearing model of ORFV120 gene deletion strain with the injection dosage of 1 multiplied by 10 ⁶ TCID ₅₀ The results are shown in FIG. 4. Can obviously reduce the weight and volume of the tumor.

5. Effect of ORFV120 gene-deleted Strain therapeutic injection on B16 tumor metastasis

The application utilizes ORFV120 gene deletion strain to inject mice lung tumor-bearing model intravenously with the injection dosage of 1 multiplied by 10 ⁶ TCID ₅₀ The lung metastasis results of the tumors are shown in fig. 5. The absence of toxins can significantly reduce the size and number of lung metastases.

The results of the intraperitoneal injection of the ORFV120 gene-deleted strain into the lung tumor-bearing model of the mice show that the effect of treating the change of the tumor amount in the lung after the lung metastasis of the tumor-bearing mice in the form of intraperitoneal injection is consistent with that of fig. 5.

ORFV120 Gene deficiency in example 2The preferred dosage of the strain loss can also be 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁷ TCID ₅₀ Can increase CD3 ⁺ T lymphocytes and CD8 ⁺ T lymphocytes significantly reduce the weight and volume of tumors, significantly reduce the size and number of lung metastases, and have biosafety.

Example 3

Example 3 the antitumor drug obtained in example 1 was used in combination with at least one of antitumor radiotherapy, antitumor chemotherapy, antitumor immunity, antitumor targeting, antitumor hormone drug. The chemical, antibody drug, targeted inhibitor and hormone drug used in combination are administered by intraperitoneal injection or oral administration, and are administered at staggered time intervals with the antitumor drug comprising the attenuated strain of the infectious impetigo virus.

Combined use with anti-tumor chemotherapy: abdominal injection of Etoposide (Etoposide is a cell cycle specific antitumor drug with molecular formula of C ₂₉ H ₃₂ O ₁₃ ) 10-50mg/kg, once every two days; the medicine for treating tumor, which comprises the attenuated strain of the sheep infectious impetigo virus, adopts the intratumoral injection mode, and is staggered and spaced with the anti-tumor chemotherapeutic medicine for administration, and the dosage is 10 ⁶ TCID ₅₀ 。

Use in combination with anti-tumor immunotherapy: PD-1 and PDL-1 antibodies are administrated by intraperitoneal injection, and 2.5-10mg/kg is administered once every 2 days according to different tumors; the medicine for treating tumor comprising the attenuated strain of the sheep infectious impetigo virus adopts the mode of intratumoral injection and is staggered and spaced with the anti-tumor immunotherapy medicine for administration, and the dosage is 10 ⁶ TCID ₅₀ 。

Use in combination with an antitumor targeting drug: the antitumor targeting medicine is administrated by intraperitoneal injection, and the PI3K alpha inhibitor BYL719 is 5-20mg/kg once every two days; the medicine for treating tumor comprising the attenuated strain of the sheep infectious impetigo virus adopts the mode of intratumoral injection and is staggered and spaced with the anti-tumor immunotherapy medicine for administration, and the dosage is 10 ⁶ TCID ₅₀ 。

Combination of anti-tumor hormone therapies: antitumor agentThe hormone therapy drug is subcutaneously injected, and tamoxifen is used for treating ER-positive breast cancer at a dosage of 10-100 micromoles/kg/day; the medicine for treating tumor comprising the attenuated strain of the sheep infectious impetigo virus adopts the mode of intratumoral injection and is staggered and spaced with the anti-tumor immunotherapy medicine for administration, and the dosage is 10 ⁶ TCID ₅₀ 。

The results show that the combination of the medicine for treating tumor comprising the attenuated strain of the sheep infectious impetigo virus and the anti-tumor radiotherapy, the anti-tumor chemotherapy, the anti-tumor immunity, the anti-tumor hormone or the anti-tumor targeting medicine can effectively inhibit the growth or the metastasis of the tumor.

Example 4

The antitumor drug containing the attenuated strain of the sheep infectious impetigo virus prepared in example 1 inhibits in-situ growth of tumors by means of intratumoral injection; inhibit tumor metastasis.

Preferably, the intratumoral injection dose is 1×10 ⁶ TCID ₅₀ The injection is carried out once every other day for 3 times. In situ grown tumors are inhibited, representing a reduction in tumor volume and weight.

Preferably, the intravenous dose is 1X 10 ⁶ TCID ₅₀ The injection is carried out once every other day for 3 times. Lung metastasis of tumors is inhibited, manifested by a reduced volume of metastases and a reduced number of metastases colonization in the lung.

Example 5

The ORFV120 gene-deleted strain prepared in example 1 was mixed with 50. Mu.g of a stabilizer to prepare an antitumor drug. The stabilizer is lactose, glycerol, sucrose, mannitol, trehalose, fructose, galactose, glucose, amino acid, dextran, polyethylene glycol, etc.

Example 6

Example 6 is similar to examples 1 to 5, except that example 6 provides a different manner of virulence gene deletion to produce an attenuated strain of the infectious impetigo virus.

As shown in SEQ NO:20, are predicted to include 4 virulence genes, respectively virulence genes ORFV001, ORFV005, ORFV007, ORFV008. The manner of preparing an attenuated strain of the infectious impetigo virus of sheep comprises the steps of: 20, and also includes a long fragment knockout covering one or several virulence genes, said long fragment referring to a nucleotide sequence of at least one virulence gene of ORFV001, ORFV005, ORFV007, ORFV008, said long fragment referring to a sequence of SEQ NO: 20.

The knockout mode adopts the conventional method of gene knockout in the prior art, such as designing homology arms at the starting and ending sites of the knocked-out gene, and knocking out or replacing the homology arms with other nucleotide sequences by homologous recombination.

SEQ NO:20, comprising 4 gene distributions of virulence gene regions: ORFV001 (nucleotide 3722 to 4171 in the nucleotide sequence of SEQ NO: 20), ORFV005 (nucleotide 5201 to 5428 in the nucleotide sequence of SEQ NO: 20), ORFV007 (nucleotide 5511 to 5990 in the nucleotide sequence of SEQ NO: 20), ORFV008 (nucleotide 6053 to 7603 in the nucleotide sequence of SEQ NO: 20).

Wherein, a preferred embodiment, the long fragment virulence gene comprises SEQ NO:20, that is to say the knockout of the entire nucleotide sequence shown in SEQ NO:20, such that all four virulence genes are deleted.

Alternatively, the co-deletion of genes that are completely discontinuous and/or not completely continuous may be used. At least any two genes of ORFV001, ORFV005, ORFV007, ORFV008 were deleted.

Still another preferred embodiment is deletion of consecutive gene segments: SEQ NO:20, from an ORFV001 to an ORFV005, from an ORFV001 to an ORFV007, and from an ORFV001 to an ORFV008; ORFV 005-ORFV 007, ORFV 005-ORFV 008; ORFV007 to ORFV008. I.e., SEQ NO:20, wherein N1 is between ORFV001 and ORFV007 and N2 is between ORFV005 and ORFV008.

Alternatively, SEQ NO:20, and replacing the sequence from the Nth 1 gene to the Nth 2 gene in the sequence shown in the specification by other nucleotide sequence fragments, such as replacing the deletion fragment by a nucleotide sequence of a marker gene (such as EGFP (enhanced green fluorescent protein) and the like). It is further preferred that the marker gene is further replaced with other exogenous functional genes for enhancing the immunostimulatory capacity.

The final mode of construction of gene deletion was selected in this example, i.e. the start site of the defined knockout sequence (i.e. deletion sequence) was located in SEQ NO:20 (4 gene fragments in total, each having the nucleotide positions set forth above) and a termination site located at the ORFV 005-ORFV 008 gene, wherein the most preferred knockout sequence is SEQ NO:20, namely the ORFV 005-ORFV 008 gene of the nucleotide sequence shown in the formula 20, namely the original gene of the knockout sequence is the ORFV005 gene, and the termination gene is the ORFV008 gene. Further preferred is to divide the sequence of SEQ NO:20, and inserting a nucleotide sequence of a marker gene after knocking out the nucleotide sequence shown in the figure. The final preferred embodiment is to further remove the nucleotide sequence inserted into the marker gene by recombinant means.

Similarly to examples 1 to 5, the virulence gene-deleted attenuated strain prepared in example 6 was used as an antitumor drug containing an attenuated strain including an infectious impetigo virus of sheep (the administration mode and the dose were the same as those of examples 1 to 5), and the proportion of T lymphocytes in the tumor was significantly increased. In particular CD3 ⁺ T lymphocyte increase and CD8 ⁺ T lymphocytes increase. The weight and volume of the tumor can be remarkably reduced by injecting the gene-deleted strain into the tumor-bearing model of the mouse in the tumor. The size and number of lung metastasis tumors of the mice are obviously reduced by intravenous injection of the gene-deleted tumor strain described in the example 6 into the lung tumor-bearing model.

Example 7

Example 7 is similar to examples 1 to 5, except that example 7 provides a different manner of virulence gene deletion to produce an attenuated strain of the infectious impetigo virus.

SEQ NO:21 comprises 22 genes of virulence gene regions: ORFV112 (SEQ NO:21 from nucleotide 124 to nucleotide 987), ORFV113 (SEQ NO: nucleotide 1060 to 1686 of nucleotide sequence No. 21), ORFV114 (nucleotide 1730 to 2770 of nucleotide sequence No. 21), ORFV115 (nucleotide 2887 to 3318 of nucleotide sequence No. 21), ORFV117 (nucleotide 3387 to 4082 of nucleotide sequence No. 21), ORFV117 (nucleotide 4257 to 5054 of nucleotide sequence No. 21), ORFV118 (nucleotide 5308 to 5616 of nucleotide sequence No. 21), ORFV119 (nucleotide 6014 to 6614 of nucleotide sequence No. 6614 of SEQ NO: 21), ORFV121 (nucleotide 7787 to 8689 of nucleotide sequence No. 21), ORFV122 (nucleotide 8743 to 9714 of nucleotide sequence No. 21), OR123 (nucleotide 135 to 12905 of nucleotide sequence No. 21), or FV 35 to 158125 of nucleotide sequence No. 35 to 12935 of nucleotide sequence No. 21, or fv125 of nucleotide sequence No. 35 to 12935 of nucleotide 15835 of nucleotide sequence No. 21, or nucleotide sequence No. 35 to 158125 of nucleotide sequence No. 21, or FV120 (nucleotide sequence No. 21 to 17580 of nucleotide sequence No. 21) ORFV130 (nucleotide 19214 to 20701 of the nucleotide sequence shown in SEQ NO: 21), ORFV131 (nucleotide 20664 to 21338 of the nucleotide sequence shown in SEQ NO: 21), ORFV132 (nucleotide 21392 to 21793 of the nucleotide sequence shown in SEQ NO: 21), ORFV134 (nucleotide 22633 to 23082 of the nucleotide sequence shown in SEQ NO: 21).

The manner of preparing an attenuated strain of the infectious impetigo virus of sheep comprises the steps of: 21, and also includes a long fragment knockout covering one or several virulence genes, said long fragment referring to a nucleotide sequence knockout of at least one virulence gene of ORFV112, ORFV113, ORFV114, ORFV115, ORFV116, ORFV117, ORFV118, ORFV119, ORFV120, ORFV121, ORFV122, ORFV123, ORFV124, ORFV125, ORFV126, ORFV127, ORFV128, ORFV129, ORFV130, ORFV131, or ORFV134, said long fragment referring to a sequence comprising a virulence gene and a spacer gene between virulence genes: 21.

In a preferred embodiment, the long fragment virulence gene comprises SEQ NO:21, deleting the entire nucleotide sequence set forth in SEQ NO:21, i.e., 22 virulence gene knockouts.

In another preferred embodiment, the long fragment of ORFV, the polygene deletion pattern comprises the sequence of SEQ NO:21, or a combination of deletions comprising any 2or more genes between the order of 2or more genes in the nucleotide sequence designated by 21, or 112, or 113, or 114, or 115, or 116, or 117, or 118, or 119, or 120, or 121, or 122, or 123, or 124, or 125, or 126, or 127, or 128, or 129, or 130, or 131, or 132, or 134, or 112 to 134, or any combination of deletions comprising either consecutive or non-consecutive genes (including complete and/or incomplete consecutive as described herein).

Another preferred embodiment is the following successive gene fragment deletions: SEQ NO:21, from ORFV112 to ORFV113, from ORFV112 to ORFV114, from ORFV112 to ORFV115, from ORFV112 to ORFV116, from ORFV112 to ORFV117, from ORFV112 to ORFV118, from ORFV112 to ORFV119, from ORFV112 to ORFV120, … …, from ORFV112 to ORFV132, and from ORFV112 to ORFV134; ORFV 113-ORFV 114, ORFV 113-ORFV 115, … …, ORFV 113-ORFV 134; ORFV 114-ORFV 115, ORFV 114-ORFV 116, … …, ORFV 114-ORFV 134; ORFV 115-ORFV 116, ORFV 115-ORFV 117, … …, ORFV 115-ORFV 134; ORFV 116-ORFV 117, ORFV 116-ORFV 118, … …, ORFV 116-ORFV 134; ORFV 117-ORFV 118, ORFV 117-ORFV 119, … …, ORFV 117-ORFV 134; ORFV 118-ORFV 119, ORFV 118-ORFV 120, … …, ORFV 118-ORFV 134; ORFV 119-ORFV 120, ORFV 119-ORFV 121, … …, ORFV 119-ORFV 134; ORFV 120-ORFV 121, ORFV 120-ORFV 122, … …, ORFV 120-ORFV 134; and so on to the ORFV 132-134. I.e., SEQ NO:21, wherein M1 is between ORFV112 and ORFV132 and M2 is between ORFV113 and ORFV134.

Another preferred variant is to divide the sequence of SEQ NO:21 to the M2 gene, and in a preferred embodiment of the present application, the deletion fragment is replaced with the nucleotide sequence of the marker gene. It is further preferred that the marker gene is further replaced with other exogenous functional genes for enhancing the immunostimulatory capacity.

The final mode of construction of gene deletion was selected in this example, i.e. the start site of the defined knockout sequence (i.e. deletion sequence) was located in SEQ NO:21 (22 gene fragments in total, each having the start and stop nucleotide positions as described above) and the termination site located in the ORFV113 to ORFV134 genes, wherein the most preferred knockout sequence is SEQ NO:21, namely the ORFV 119-ORFV 125 gene of which the initial gene is the ORFV119 gene and the termination gene is the ORFV125 gene. Further preferred is to divide the sequence of SEQ NO:21, and inserting a nucleotide sequence of a marker gene after knocking out the nucleotide sequence shown in 21. The final preferred embodiment is to further remove the nucleotide sequence inserted into the marker gene by recombinant means.

Similar to examples 1 to 5, the virulence gene-deleted attenuated strain prepared in example 7 was used as an antitumor drug containing an attenuated strain including an infectious impetigo virus of sheep (the administration mode and the dose were the same as those of examples 1 to 5), and the proportion of T lymphocytes in tumors, particularly CD3, was significantly increased ⁺ T lymphocyte increase and CD8 ⁺ T lymphocytes increase. The weight and volume of the tumor can be remarkably reduced by injecting the gene-deleted strain into the tumor-bearing model of the mouse. The size and number of lung metastasis tumors of the mice were significantly reduced by intravenous injection of the gene-deleted tumor strain described in example 7 into the lung tumor-bearing model.

Example 8

Example 8 is similar to examples 1 to 5, except that example 8 provides a different manner of virulence gene deletion to produce an attenuated strain of the infectious impetigo virus. Setting SEQ NO:20 (at least one of ORFV001, ORFV005, ORFV007, ORFV 008) and the nucleotide sequence of SEQ NO:21 (at least one of the nucleotide sequences of ORFV112, ORFV113, ORFV114, ORFV115, ORFV116, ORFV117, ORFV118, ORFV119, ORFV120, ORFV121, ORFV122, ORFV123, ORFV124, ORFV125, ORFV126, ORFV127, ORFV128, ORFV129, ORFV130, ORFV131, and ORFV 134) and simultaneously performing deletion mutation, to prepare an antitumor drug comprising an attenuated strain of an infectious impetigo sheep virus. The results show that the anti-tumor drug provided in the example 8 can increase the proportion of T lymphocytes in tumors and inhibit tumor growth.

Example 9

Example 9 is similar to examples 1 to 8 except that the deletion sequence does not include the nucleotide sequence of the ORFV132 gene.

Sequence listing

<110> Jilin university

<120> a medicament for tumor treatment

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 588

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgcgtctaa tcttagcgct cgtggcttgc ttgttggcgg cgccgatgcc gttatcgggt 60

cgttcgacaa gcactccaaa cacgtccccc tccgtactcg gctcgacgag ttcggaacca 120

agctcggaag acgctgtggc ttcgagcaca actacaagca cattcacaag cacactcact 180

ctgtccacaa gtgtggacac cactactacc tcgggcgcta caacgtccgc aaacagcact 240

cctgcagcga gtgtgagctc ctccacaccc gcaaccaccg aggcatcgac ggcaccaacg 300

acgccgtcga cgccgacgac agtgaagggc aaggaagacg cgaaggcgtc tgcctacctc 360

gctttactaa tcacgttcat ggtcatgacc acgctagtga tggtcgtggt cgtggtcgtg 420

gtcgtgtaca aacagggact ctgtgactgc tgctgtagga tgtttccctg ctgcagagag 480

ctcaaggact acctcgacga ggaggagagc gccggtctgt acgacgcctt gacgtggagc 540

cactcgaacc ccggcttccg gctcgtcatg cgcgcggacc ccagatga 588

<210> 2

<211> 391

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ccccctccgt actcggctcg acgagttcgg aaccaagctc ggaagacgct gtggcttcga 60

gcacaactac aagcacattc acaagcacac tcactctgtc cacaagtgtg gacaccacta 120

ctacctcggg cgctacaacg tccgcaaaca gcactcctgc agcgagtgtg agctcctcca 180

cacccgcaac caccgaggca tcgacggcac caacgacgcc gtcgacgccg acgacagtga 240

agggcaagga agacgcgaag gcgtctgcct acctcgcttt actaatcacg ttcatggtca 300

tgaccacgct agtgatggtc gtggtcgtgg tcgtggtcgt gtacaaacag ggactctgtg 360

actgctgctg taggatgttt ccctgctgca g 391

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gtcgacatct atatactata tag 23

<210> 4

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctcctcgccc ttgctcacca tggtggcgaa ttcctcgagg cgtc 44

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggtgagca agggcgagga gctg 24

<210> 6

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aagcttagat ctgcggccgc ttacttgtac agctcg 36

<210> 7

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gtgaattcga gctcggtacc aagcttgccg cacccccgca gccccag 47

<210> 8

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atatagtata tagatgtcga cacgtgtttg gagtgcttgt cgaacg 46

<210> 9

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gagctgtaca agtaagcggc cgcagagctc aaggactacc tcgacg 46

<210> 10

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

accatgatta cgccaagctt agatcttcag gcagtcgttc atgga 45

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gctcgacgag ttcggaacc 19

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gcagtcacag agtccctg 18

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

atggtgagca agggcgag 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ttacttgtac agctcgtc 18

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gctcgacgag ttcggaacc 19

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gcagtcacag agtccctg 18

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

atggcctcct ccgaggacgt ca 22

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ttaggcgccg gtggagtggc gg 22

<210> 19

<211> 460

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cgccgatgcc gttatcgggt cgttcgacaa gcactccaaa cacgtccccc tccgtactcg 60

gctcgacgag ttcggaacca agctcggaag acgctgtggc ttcgagcaca actacaagca 120

cattcacaag cacactcact ctgtccacaa gtgtggacac cactactacc tcgggcgcta 180

caacgtccgc aaacagcact cctgcagcga gtgtgagctc ctccacaccc gcaaccaccg 240

aggcatcgac ggcaccaacg acgccgtcga cgccgacgac agtgaagggc aaggaagacg 300

cgaaggcgtc tgcctacctc gctttactaa tcacgttcat ggtcatgacc acgctagtga 360

tggtcgtggt cgtggtcgtg gtcgtgtaca aacagggact ctgtgactgc tgctgtagga 420

tgtttccctg ctgcagagag ctcaaggact acctcgacga 460

<210> 20

<211> 7770

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

aacaagttgt cggttaactc actctctcac tcggattaag agaacaagtg tcgttaactc 60

acactcactc tctcacctga gtaagagaac acgtcaacag tcaactcact caagtaacgg 120

gtaggtggcg agagaacaac tcactcggaa ggagagtgga cgacgagaga acgggctaac 180

ggagtaactc tcgcccactc gcccactcac ccgctagagt aggcaacgag aacgcgttag 240

ccgcggaccc tcgacccctc gctagacttt agcgggacgg tcggccctcc aaactcctgt 300

aaagttttcg gaggccgtag tctccctcga aaactcttcg taaaaacctt ttcgcggaac 360

ctcgagggta gcgacctctc ttaaaacttt acgaggtcgg tcttcctctc gaaaactttt 420

gtaaagactg gaggcaggac ctccccgaaa actttcgtaa acacttttcg gaggagcagc 480

ttcctctctc caaaacttta ggaggacaag agacctcctc gaacactttt cgtaaagctc 540

ttcgcggacg gacggccgct cgcgacacgc gggcggcgga acctcggagg gcaggtcggc 600

ctctcaaaat tttgtaagac tcttccaaac ggtcgacctc ccccaaacgt ttcgtaaact 660

tttcgcggaa cctctggagg taggtcggcc tctcgaaaac tttgtaaaga ctggggtcag 720

tcgacctccc cgaaaacttt tgtaaagagt tttcgcggaa cctctggagg gtaaggcagc 780

ctctccctag acctctccaa aactttttgt aaagactttt cgtggtcagt cgacctccct 840

cgaaaacttt ttgtaaaact ttttcgcaca ccctccggga gacgggccgc cgccgcgacc 900

gcggggagcg gagagcccga ccgcgcgagg acttctcgcg ttaccgttag gtaggtcggc 960

ctctcgaaaa ctttttaaga acttttgtag acgcgttagc gggaccgtcg cgggcgcgcg 1020

gccgcccgcg acccgcgagc gggagggacc gttggaagac aggtccgcga aaacttttag 1080

cggagcctct ggaaggtagg tcaccctctc gaaaactttt tgtaaacctt tagcggcccg 1140

tcgcgggcga gcggcctccg aggcacgcga gcgggaggga ccgttggaag acaggtcggc 1200

ctccctcgaa aactttttat aaaactttta taaaaacttt tacgcggacc cgttggaagg 1260

taggtcggcc tccctcgaag tctaaaaact ttttcgcggg actcggacgg cactgtcacc 1320

cgactcctga cttcttactc ctcgacttct gtctccctgt ctcccggact cctgactcct 1380

tgacttctaa agcgaggtct cgcggctgcg gagtgccgcc tccgcggagt cggagtcctc 1440

ctccgcggag tcgcgtttct gcggacgccc gtcctcaagg gcattcagca gttccagcct 1500

ccgctgtaac tcctcccgca ggagctcctg gtccgcgttc tcgcggcagc gcggctcagc 1560

cgccgcggga gccggccgcc gccccggaga agccgcggat ccgctgttcc ggcgaaggca 1620

ggacagacat ttttttcggc ccataaatta aaaagaatca acagaaaatt atcatgatga 1680

taaaaacttt taggattgta tttagttgaa cgtttcgtca ataaatcaac ctcaaatttt 1740

ttcacatgta atttcttgtt ctttttaatt tatgggccga aaaaaatgtc tgtcctgcct 1800

tcgccggaac agcggatccg cggcttctcc ggggcggcgg ccggctcccg cggcggctga 1860

gccgcgctgc cgcgagaacg cggaccagga gctcctgcgg gaggagttac agcggaggct 1920

ggaactgctg aatgcccttg aggacgggcg tccgcagaaa cgcgactccg cggaggcggc 1980

actccgcagc cgcgagacct cgctttagaa gtcaaggagt caggagtccg ggagacaggg 2040

agacagaagt cgaggagtaa gaagtcagga gtcgggtgac agtgccgtcc gagtcccgcg 2100

aaaaagtttt tagacttcga gggaggccga cctaccttcc aacgggtccg cgtaaaagtt 2160

tttataaaag ttttataaaa agttttcgag ggaggccgac ctgtcttcca acggtccctc 2220

ccgctcgcgt gcctcggagg ccgctcgccc gcgacgggcc gctaaaggtt tacaaaaagt 2280

tttcgagagg gtgacctacc ttccagaggc tccgctaaaa gttttcgcgg acctgtcttc 2340

caacggtccc tcccgctcgc gggtcgcggg cggccgcgcg cccgcgacgg tcccgctaac 2400

gcgtctacaa aagttcttaa aaagttttcg agaggccgac ctacctaacg gtaacgcgag 2460

aagtcctcgc gcggtcgggc tctccgctcc ccgcggtcgc ggcggcggcc cgtctcccgg 2520

agggtgtgcg aaaaagtttt acaaaaagtt ttcgagggag gtcgactgac cacgaaaagt 2580

ctttacaaaa agttttggag aggtctaggg agaggctgcc ttaccctcca gaggttccgc 2640

gaaaactctt tacaaaagtt ttcggggagg tcgactgacc ccagtcttta caaagttttc 2700

gagaggccga cctacctcca gaggttccgc gaaaagttta cgaaacgttt gggggaggtc 2760

gaccgtttgg aagagtctta caaaattttg agaggccgac ctgccctccg aggttccgcc 2820

gcccgcgtgt cgcgagcggc cgtccgtccg cgaagagctt tacgaaaagt gttcgaggag 2880

gtctcttgtc ctcctaaagt tttggagaga ggaagctgct cctccgaaaa gtgtttacga 2940

aagttttcgg ggaggtcctg cctccagtct ttacaaaagt tttcgagagg aagaccgacc 3000

tcgtaaagtt ttaagagagg tcgctaccct cgaggttccg cgaaaaggtt tttacgaaga 3060

gttttcgagg gagactacgg cctccgaaaa ctttacagga gtttggaggg ccgaccgtcc 3120

cgctaaagtc tagcgagggg tcgagggtcc gcggctaacg cgttctcgtt gcctactcta 3180

gcgggtgagt gggcgagtgg gcgagagtta ctccgttagc ccgttctctc gtcgtccact 3240

ctccttccga gtgagttgtt ctctcgccac ctacccgtta cttgagtgag ttgactgttg 3300

acgtgttctc ttactcaggt gagagagtga gtgtgagtta acgacacttg ttctcttaat 3360

ccgagtgaga gagtgagtta accgacaact tgttgactcg ttgacttgtt atctcgccat 3420

ccactctcgc tcgagcgagt gagtgctgtg ttagcggtca tccgttctct cgttctctct 3480

cttactccga gtgagtgagt gtgtgagtta accgttaacc gttaactcgt tatctagtta 3540

acttgttctc ctactctgag cgagagagcg agtcacggtt aactgttcct tacccgagcg 3600

ggcgggcgat aaaaataatt atattattcg tccgtccacg aggcgaagga agggcggcga 3660

ggggatgctg gtctaatcta ctaaggccga ttacaaaaac ggatgggaga ccggggaggg 3720

gtcacagctc cgagcggtgc atccgcgcca gctggcggcg ccactcggcc gcgggccgcg 3780

cggccgccca ggccgcgttg tagccgcccg ccaccgcgac gcaggtcagc tcgaactccg 3840

gcccgagctc gcgcacgtcg tagatgtgca cggtcgcgac gtgcagcagc agcgcgccct 3900

tgcgcgcgga ggagaagctc gcgtgcatgc cggcggagag cgggtacgcc ggcgagagcg 3960

tcccgccgcc gtgcgcgacc accgccatgt gccgcccgtc gccgccgacg gtcaggcagg 4020

tcgccgaggc ggagccgttc gcgcggacgg ggcccgcctc caggaaggcg gccggcagcg 4080

gcggcgccgc ggggcggaag agcccgccga ggaggaagcc cagcgccacc agcgcgagcg 4140

cgccgagcag cctgcgcggc gaggggtgca tgctgtgtcg gctgtggtgc ggagggcggg 4200

tgagaggggc gtccgcggcc gtggcgggag ggtctcgact gctaggcggt cctttttcac 4260

tttgcgccgt ggcgcctcct tccgcatgac ctggtcgcat cattgtttgg atctgcggtc 4320

cataaaaagt tgctagcggc gtcttctggg cggccttgtg gtcgccgccc ccgcgcgccc 4380

gccgcgcgca gcagcgccgc cgccagcagc gcgcggacca gtgccggcga ggggcgccgc 4440

gcgcggatca ggtactccac gagcagggtg gtgagcaagg attctcgaga agtaggagtc 4500

atgtgtgacg acaaggagaa acgttatatt aggcgcgtcc tacttcattt tgaagatggg 4560

tagaagtgtt aaaaacttaa caccactgtt cactccacca ctgccgttgc tgtgtcctgc 4620

cccaggcgac tacagtgctt tttccaccgc ttgttccaaa agctccccat ccattgttgt 4680

tagaactttc agatgtttgt ttatctaggt cgtttagttc caccgcgaga gttttgacca 4740

ttatcgtttt ggacatgctg ttggtaataa gtttaataac caattataaa aatagttata 4800

atgtataatt tgttatatat gctaataagg taacaaacac tttaatgtta tattttgcct 4860

atccctccgt taataccacc attaacacca ccattaacac cggagctttt accaccaccc 4920

caaaagccac cacattgtct cttgccacca ccgatccctg ttccacccca aaaagcaccc 4980

catccactgt taccaccaca agaactctta tctattttgc tgggtgtttg agttatctct 5040

ccatatggtc ttttaatacc acctgcgttt tcagacattg tttgtaataa taagttaatt 5100

aacaataaat ttagacactg tgtctcacgt cttaaaaata aaacgcggtt acaatttaca 5160

atttaaacaa taaatagcag caattcgtat ataaaaatag ttatatttta ccacacccac 5220

cattaacacc accattaaca ccaccactta agcttttacc accacccaaa aatccaccat 5280

tactacctcc accaacacac attcttttct ctaaaggtcc ccaaagtcca cctgaacttg 5340

gacgttttac agcacctccg ggtgtacttg cgtacccttt agaagttcca ccgtgactgt 5400

agatatgata ctgtccttgt ccaggcatga ttaaagtgtg ttgaattagt gctatctaca 5460

caactgtgcg agacgctcga ataaaaagaa gctacatttt acaattttga ttagctgatg 5520

taccacgctg tatcgcggcc accacaagca cccgatccag tagaaccaaa tccagagtcg 5580

ccgcggtcgg tgttgtccaa gctgttaact tcttgaactg ctgggcacga tatgcgttcg 5640

catattagct gagctatcct gtctcccttc ttaacctcaa agtcgctgtt cccgaagttg 5700

aacagcacca ctccgacatt gcctcggtag tcttcgtcga tcacgccagc gcccacgtcg 5760

atgaagtgtt tgactgcgag gctagaacgt ggtgctatgc gtccgtagca accaggtggg 5820

agctttatca gcaggtcagt aaacactacg cgactgcaat gcggagggat gacacagtcg 5880

tatgcactac ataagtctaa tcctgcggca ccaggagatc ctctggctgg tatagtggcg 5940

ttttggctga ggcggacaac ctgaagggtt tccgtgtggc agaactccat ggctagggtg 6000

gcgagcggcc gatcgactac ggggtgtaca atttacactt tcttcagaaa aatcaggggt 6060

tggtcagcat ggcgcggcgc aggtccagca gcgagtcgta cgacaggaag cacaggatgg 6120

aggtcacgat ctccggcggc agggcgcacg ggcacatgag gccagcgatc tgctcggcca 6180

gcgagacgcg cagccgcatc atgcagatct tgctgaagag cgccgtcccg tagatgggga 6240

acccggccgc gcgctccagg aaggcgttcg acacgaagag cgccttcgcg tcgtccgcag 6300

cgcgcagcac gtccagcagc gtcgcgtccg tgtggcagcg caccgcgcgc atgctcgcga 6360

tctcctgctc gcacgagcgg atcacggttg cgtagtccac cagcgcgcgc tccgctagca 6420

tgcgcgcgcc ctcgccgcgc agcgccagct cctgcacgca cagcagcgcg gcctccgagc 6480

gtcggaacac gtggccccat tgctcggatg tgatcagcgc gcgcgcgagc agctccgtcg 6540

gcgggcggcg cgcgagcacg gccgccgtcg cgcgcacgtt gttgcggcgc agcatctctg 6600

agaccgcgca taagcccgag gccgccacgt gctcgagctc cgcgcccatg cgcaccagcc 6660

ggcagcaggc gccgtggctg aacaccgccg cgcggtgcag cgcggtctgc tggttgttgt 6720

tgcgcaggtt caggtccagc ccgcgctcga gcacaaagtc cacaatgccg cgctcgcagc 6780

gcccgtaggt cgccatgtag tgcagcatgg tgttcccgca cgcgtctacg gcggccgggt 6840

ccacgcccag gcccgtgagc gtgcgcacaa ggacttcgga aatcttggcc gtgcgcgcga 6900

ggtggtgcag tgtcgtgcgc tcgtacgcgt ccacgacgcg cgcgtctgcg cccgcgcgca 6960

gcatcacgtc cacgagcgcg gcggagacgc cgccggagca cagcagcgcc gccagcggcg 7020

tcaagccgtg acagtcgcag gcgtttgggt tcgcgccgcg ctcgagcagc agccgcagca 7080

cgtcctcgcg gatccactgg ttcttggcgt acacgtgcag cggcgttacg ccgtaggtgt 7140

tgccctcgtt cacgcgcgcg cccgcgtcca gcagcagccg cgcaacctcg agctcggcgc 7200

cgtcggggcc gcagaaagcc aggaaggagg agagcacgct gtcgcagacg acgaagctgg 7260

cgtcgccgac cacgtccgcg cctgcctcca gcatgagcgc gaccacctcc ggccgcacgc 7320

agtcgtactg cacgtaggcg tgcagcggcg tgcggccgca ggagtccttg gctttcacgt 7380

ccgcaccggc ctccagcagc acgcgcacga tctccgcaca ctgctcgtgc cgcgcgaagt 7440

gcacgcagag gtgcagcggc gtgcgctcgt gctcgccgcg gaagttcacg tccgcgtcgg 7500

tggccacgag cgcgcggacc gtttcgaggt ccacctgccc tgactccagg tagcggaaga 7560

gcaggtccgc gtgcgggacc acgacggact cccgcgagag catggctgcg gcgtctacaa 7620

atattgaaat cttttttcac tcatctttat gggcgctgaa cgcgcaataa gggtgggagt 7680

aaaaaacttt tacaaaaagc gtacaaaagg tacaaaaggt aaaaaaggta caaaaggtag 7740

aaaaggcggg gcggggacgg gctggcaggt 7770

<210> 21

<211> 23240

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gctggacgta accatataat cagcatcctt gtttttatcc tgtcttttta tcagtttttt 60

agctagttaa aacataaata gtaaagctaa aaagaggagt tctggagtct tgtaacaacc 120

aggatgaagg cggtgttgtt gctggcgtta ctgggagcgt tcactaacgc agcgcccgtc 180

ggcaaccagc gtctcgatag tggggagaaa gaaaaattct gctcgacgca tcaggatgaa 240

gtgtacgcta ggttcaggct tcagatgcgc gtgggtgtac gacacagtcc tttctacact 300

cccagcaaca tgtgcatgat ggacatagaa gactccgttg atgacataga agaagactcc 360

attatagtaa aagaatttac gtctacggcc acgggtgagg cggacggagt gaacgtgtcc 420

gtggcactag tgggagaaga cgtgaaaata ccgttaagtt acataggcct tggattcaac 480

ccatctacag atggctacct gtacgtcaac gtctcgtcac gagctccttg ggttcaacag 540

actctagacc tatctgcgaa cgacagctgg ggtattaaac aggttctaga aaaagagtta 600

ctggccatcc aaatagggtg cgacaaccaa aaatttcctg aagaacccac aaccacccaa 660

cctccctcac ttgtcacgac aacgctttcc tcaacaactc cagatctgaa tgacgaaaac 720

acagacacta cgccgacgac cactggcgcc agtgtagaca gaaagcgcaa tccagctgac 780

tttagtttct cgctgctcgt ggacccccga tgcgtgactt ctgtagacct gcacgtcgag 840

ctcagggacg cgtgcatgga gtacaaagaa acgtcgccgt tgtcgctgaa gggggaatat 900

ggagacggcg aactagtaaa aaaggagatc aaagacgtgg gaaagaatca caatatgtgc 960

agtcttaacc tcaaccctgg caattaagct gtttttattc ggcaatataa taggtgatta 1020

ttgaacatta aacaaaactt atcccacaac gccgcaacaa tggaagtgct ggtgatcatc 1080

tctattatcg tcgccgtaat atgcctaacc ggagcggtga tgtacctcct tattgaactc 1140

ggcttagccg ccgagcgcgc taacaaacgc gcgcgcgtga agaaaaatat gcgcaaatta 1200

gccactcaat tgggaaatgg atctgttgaa tccggcatag gcccgtgcac aatatcgcgc 1260

accatggact ttggacctag tcgctgggac agcgacagtg agggtgacgg agacagcatg 1320

tccacgacgt ccaccagcgg aggggggact ctcccccgag tgtgggtggg gggcgggccc 1380

gggcccatgt acgaaaactt ctgcgggaac ggcacccgcc actcccccac caacgaccct 1440

ggctaccact cgcgggagac tctctgcagc ggacctcccc gtcaggcgcc ggatctaccg 1500

cccaccccga agcccgacga ggtagaggtg gaagtggggc ccggttccga cgaccaacaa 1560

ggtccgtacg aggaacctga tcccattctc ccgcaggaac ccgagccgcc ggtgcagatt 1620

gaggtaacca tcaacgggcc cggcggagaa ggcgaggcgg agggagaatt tttctacgac 1680

gagtagccgc caaaactgaa taactatcgg gcttcgtaaa cgcgcagaca tgccgctgtt 1740

ccggaagctc atggtttcgc gctccctggt caaggaatgt ctgactctgg acttccggca 1800

gggcgagcgt ctgcccacgc gatgcttcct cccggtgccc gcggggacga cattccacag 1860

agtctgcgac acctcgccgc tgacgaacga agtctcccgg cacgtgcagg agcccgtcat 1920

gggcaccgga cgggtccagt actactactt cgagagcggc cagggcatga tcggcgacaa 1980

cgcgggcatg ccgcgcatgc tcgtgtgtac gcgatcggcg tacaacggcg gcgacgtcgt 2040

cgtgcggtcc acgcggagca gagcggacaa gaccgtggtc gcgccctgcc agggcatggt 2100

gctgctgctg agccccttct gcgccttcga catcacgccg gtggagagcg gctccgcgat 2160

attcgcggag gtcatcgtca ccgcgcccag catggaccac gtcgaggcgg tcaccggcac 2220

gggcgaggcg gccgtgcgga tattcaactc gcaccacccg ctctggccgc gacacggctc 2280

gaacgtctgc ttcgcgctgc ggttgctgcg agacgtgcgc acgggcgagc gcgtggtcga 2340

gcagatgttc atggacgggc gctggcacac cgtgctgagg acgtcctgcg gcaacaaggt 2400

ctgcgtgccc gccgacctcg tgggccagac gaacctcgag gaggtgccct tctgcgacgt 2460

gacgcccgag atcatgcgcc gcgcgctggc gatcgacccg ccgtacgagg ccgtggcgca 2520

cccgcaccgc tgcgtgtacg gcgccatgga cgtccggtgc gcgaacgagt acctcgtgta 2580

ctgcaccttc aagacggagc cggcgcggcg cagcacgtcc tcgccgggcc cggacggccc 2640

cctgtcgccc gcaactccgt cgacctcgcg ggccgcggcc gcgcgcgccc cgacgacgcc 2700

acaggaggtg acctcgccaa ccacgaggct cgtggagacc tgtctgcgcg acgccctcga 2760

cggactctga cccgaaggac cccacctcac tcacgcacca cttccagacg acagactcag 2820

gctttttctg catctacctc actgataatt gaattgttat aggacaaaca ggtgcactcg 2880

agcacaatgg cgtgttttct cgaattgtta gactccatct tcaaccgacg ccaccgtaat 2940

ttcaggccgg aagacatgta caggccctct gacgccccgc cccccaaatc tcacacgcct 3000

cgcactcccc gcaccccgcg gacccagtgt cccggacacc cgcggcgaca aatctcctct 3060

cccatctacg gtgctcatgt ggactccctg ccgaggaaca gaaagcggtt ccggcatcaa 3120

cacagttgtc ccgcagatta cgagcggtgt caactcccgg acaccatcag cctggaggcg 3180

acgctgctca cggttacaat gacctccatc tccagcatat ccagttctag tagctcagac 3240

tctagctctt tggggcagtg cagactgtcc atggtgtccg cggcatcgac atccacgacc 3300

ttctcctcct cggaatgagc gccacactta tttttgtata atagtttgta ttgaacctta 3360

gagacatcca caaatagtta ggaagcatga gtagttcaag tagcgagacc acgcccaagc 3420

ctatccctgc tcctcccatg acccaggagg agtttaacaa agaagtagag aaacgggaag 3480

aaaggaaaag ggaaaaatct agaaccgttg aacgtgagtc agaaaccgta actgtatctt 3540

ccgacggatc agagaaaaca aggatctacg agcgcgagtc tgaaagaaca accgaaacag 3600

aaaagaacaa cagtccgtca actagtgata ataagcagaa cacccctgta gagaaaccag 3660

aggaaactaa gcctgcttct actcctgaag gtgagaagcc agccgagact cctgccccga 3720

ccactagccc ccaacccact acacaaccac ccgcagaatc aagccctgga agtcaaccca 3780

cacctgctcc agaaccaacc cccgcacctg agcctgcacc ggaacccact cctgccgctc 3840

agcctgcatc agtaactcaa cccgctccaa caccagagcc aagtccagcc cctgagacta 3900

ctccggcttc cgaaccaacc ccagcatctg agcctactcc tgctccagaa cctacaccat 3960

ccacaaaacc gactcctcaa ccaaccgtag aaacaccacc atctgctaca gcaccgacta 4020

ccgaagccca gccaaccaac aattccacca ctgaaactac cactggcacc agcacctcct 4080

aagtgagacc taagcatttc agagtaacgt cgtagtaagc gctagtccgc cgcgagcggt 4140

tctcgcaagt ttttttcggg taaaaagcgt acaccgtcgc cttgtcgcgg cggtgtacgc 4200

ttttttcacg ccctttttgc aaaatttaaa ttgtacccgc gccggctcta ggaaagatgg 4260

cgtgcctcag agtgctcctg gcggtgctcg cgctgtgcgg gagcgtgcgc tcggcgcaat 4320

ggatcggcga gcgcgacttc tgcacggccc acgcgcagga cgtcttcgcg cggctgcagg 4380

tgtggatgcg catcgaccgg aacgtgaccg ccgcggacaa cagctcggct tgcgcactgg 4440

cgatagaaac gccgccgagc aacttcgacg cggacgtcta cgtcgccgcg gccggcataa 4500

acgtcagcgt gtctgcgatc aactgcggct tcttcaacat gcgccaggta gagacgacgt 4560

acaacacggc acgccggcag atgtacgtgt acatggacac ctgggacccc tgggtgctcg 4620

acgaccccca gccgctcttc agccaggagc acgaaaacga aacgctgccg tacctgctgg 4680

aggttctgga gctggcgagg ctgtacattc gcgtgggctg cacggtgccc ggagagcagc 4740

cctttgaggt gatcccgggg atcgactacc cccacaccgg catggagttt ctccagcacg 4800

ttctacggcc aaaccgccgg ttcgctccgg cgaagctgca catggacctc gaggtggacc 4860

accggtgcgt gagcgccgtc cacgtgaagg cgttcctgca ggacgcctgt agcgcccgca 4920

aggcgcggac gccactctac ttcgcggggc atggctgcaa ccatccagat cgccggccaa 4980

aaaacccagt accgcgccct cagcacgtat cgtcgccgat ctccaggaag tgcagcatgc 5040

agacggcgcg ctgagggcgc tcaccgcgct gacggcggcc gtggtgtgcg cgatcgccgt 5100

cgcgctcgag cgcggggcgg aggccgacgc cgtggacctc atccttgtaa aattttcaat 5160

gatatgctag tttttatgcg accttcctta gaaaattcgg aattcaaaaa tgaaataaaa 5220

cggcgtttag cacgcatatt attaataccg actaccatgg caggcgtccg cagctgccag 5280

aagaaagtcc cttctactgc gggctccatg tcatttcaac ggggcaaccg gagcatccgg 5340

ccggcgatgt ccgaggcgct gcagaatgat ttcagctaca acccgcgacc gcctccgccg 5400

agcgcagaag agattgactt cttctgcgtg gacatgcgca aagtactgat ggaaattgag 5460

gccaagccca gcagctccaa gtaccccgat ttcatccacc cggtcgacag cagcccgccg 5520

tgcacgccgg cgcgcaagcg caacggcttc gtccgcaagg caccgaacaa aacccaggtg 5580

ccgcagcagg ccaagcgtga cggctactcc tgctaatgca gtccacacac ttcacacact 5640

aagcactcaa gctcatgatc atcacacgat gaagcagccc accagtgcca agcagcacat 5700

aaaatcaccc acttgtcctg atcgttccca attactccca atccccgtgt cttacacgca 5760

cgttaatcac cctctccttc cttcatgcgt tcctgatcgc tcctcctcct taattacaca 5820

ccccgtaatt ttgtactttt gtactttaat ttgtacactt tacacactgc ctttgtactg 5880

tatttttgta ctgaaattgg acgatattat ctttgtattc acatccaaat tttgcaaatt 5940

ccaacaccgg ttgcgaaaag tgaaatcgta ccgttttagg cttcgatccc ttcctgcgcg 6000

aagactcgcc aggatggact ctcgtaggct cgcccttgcc gtcgccttcg gaggcgtgct 6060

cgccagcatg acacagcgcc gccgcctggc ttctctcatc gccagcatcg gccaacggct 6120

gatgggcggc aacggcatgc gtcgcgtcgc cgttcggctg atcgaccagc tcatggccgg 6180

acccccggac atcaacgacg aggccttcca gcgcaagatc cgcgtgggcg tgggcgagct 6240

cttccaggcg ctccaccgcg tggtcgagca gacacgccga gagaagtact tcgaggtctg 6300

cggcgccggc aacggcgccg acgcgcccgt cgtcgagatg gacaccgcgg ccgcaccccc 6360

gcagccccag cctgctcccc tcgtgatcac gccgcagaac gcgttcatgt tcgtgccgca 6420

aggcagccac gtgcacgtgg acgagagcgt ggacccgttc ttcggcatga gcccctccat 6480

cttcgggcgc gacctccccc ctcagccgcc cgaggagctg ctgagcgact acgacccgct 6540

catgagccag gccggcgagc cgccgagccc gcggtcgccc tgcgaggccg acctctggtg 6600

cttcgagacg ctcggcgaca gcgacagcga ttgagcccgc accccaccca ccccacactc 6660

cactccttac cccacacccc aacaccccaa ccggacaatg aaggagtccc acatttcact 6720

gaaggacgcg gatgaagccg cgcatcccca catgaaggat tggcaacggt caaacatttc 6780

acctgcaatg aaggacgatg cgcggtcgcg attagcctgc gaccgacatc gcacacaaat 6840

gaaggacaca attggtttgt aatccggaca atgaagtaca aattgttttt gttaatcagg 6900

acaattggaa cacaatcaaa ttaatttttt gtacgatcat aaaatcgata tttgatgcac 6960

atatattagt aagtatatta aactaattct ccggggaggc aagcagttgg atacggcggg 7020

gcggggcacg acgtgcacgg agaattcggg cgggtccccc ttcccccacc cccacgcacc 7080

acgatgcgtc taatcttagc gctcgtggct tgcttgttgg cggcgccgat gccgttatcg 7140

ggtcgttcga caagcactcc aaacacgtcc ccctccgtac tcggctcgac gagttcggaa 7200

ccaagctcgg aagacgctgt ggcttcgagc acaactacaa gcacattcac aagcacactc 7260

actctgtcca caagtgtgga caccactact acctcgggcg ctacaacgtc cgcaaacagc 7320

actcctgcag cgagtgtgag ctcctccaca cccgcaacca ccgaggcatc gacggcacca 7380

acgacgccgt cgacgccgac gacagtgaag ggcaaggaag acgcgaaggc gtctgcctac 7440

ctcgctttac taatcacgtt catggtcatg accacgctag tgatggtcgt ggtcgtggtc 7500

gtggtcgtgt acaaacaggg actctgtgac tgctgctgta ggatgtttcc ctgctgcaga 7560

gagctcaagg actacctcga cgaggaggag agcgccggtc tgtacgacgc cttgacgtgg 7620

agccactcga accccggctt ccggctcgtc atgcgcgcgg accccagatg atgaggatca 7680

gattggcgtg tttttcccgc ccgtcgcgaa cattatgcct ctaaatgccg agaattaact 7740

gaaattcaaa cacgctttgg gactcaactc tgtggcccac acaaccatgg ctggcttcct 7800

aggcgcgttc agaggcgtgt gctccgactt atggcagtcg ctccgtggac acggcaacca 7860

ctcttccaac tgcccgcgac gacgcgccaa cagcatggac gaccgcgacc ggcgccgaca 7920

ccgccaccgc gagatcccca acagctcggc gtcgctgaac agcgacccga tgccgcaaca 7980

cagtgcgggt gcgcgccggc actacgacca ccgcccctcg gaaaagagca aacactcctc 8040

caggcaccac tctgcggacc gacaccaatc ggcggacagg gacagacacc gtcgcggtcg 8100

caagaactac gactcgcacc cgtcgcgcag gaaccgcgac tacgagcggg cggactacca 8160

gagacacccc tctcaaaccc acccagacgc ccccgcgcag acctcgacgc tcaaggtgac 8220

ctccctcagc accggctgca gcaccctgtc ccaacatcac tacgagaccc ccgaccacat 8280

ctacgacgtc ccggaagaca gtcgcggggc gtcggctccc cctcgcgcgg acctcgcgct 8340

tcccccgctc gccatgccaa aatccaagca cccgcgccgc atgcgcccgg cgtccatgaa 8400

cgactgcctg atgaagcact gcggcgccgg cagacccaac ctccaagacg acatatgcac 8460

actatgcact gatatagaga cacagctgag cgcactagag aagtctctgg agtcagagat 8520

caacttctat cgtcgctaca tacaagacac taagacattg ctcgccactc gagcagcaaa 8580

catcggcagc aaagctctga tctacaccga cgactacagc gacagtggca acgtcggcga 8640

aaaggagcac tgctcggagg agtgctgcaa agtggaggaa gttctgtgag aaagtgcgtt 8700

tttctgtaat gtgaaataag atagccttat gtgtgcacag acatggcgaa caggcttgtg 8760

tttctcgacc ccgagaccct agccgaggcc gacggcatcc ccggccaggg ggtgttcgag 8820

cccggcaaga agaaatgcaa cttcacgaag attcgcacca gcgtcgcgct cgcgtgccgg 8880

tacgccgtct cggacggcgg cctcatcgac gagttcgtca tggcgacata cgggaccaga 8940

cgcgcgtgcc ggctcgtccg gcacctgacg ataagcgcgg agggcgtgat gacccggccc 9000

gccaacaact gcgcgccgca catggtgctc atctgcctca gaggcgtggc cgccgtgtcc 9060

agcgagggca tgggcttcgg tcgctgcatc atggagcgcg gcaccatgtt catggtcaag 9120

tccgcgcaca gcgccgtcgt ctgcggcaac cccgcctgcg agctgctcgt cctcttctac 9180

gactacttca cccccatccc ccggccgctc tccggagacg aggtgctgtt tacccgcgac 9240

ctcgcgcacg tggactacct ccccgagtcg gcggtcgtct tcaagatgga ttacaacctc 9300

gagaccgacg tggccacgct gtttgtcggg gggtacatat tccgcgccaa gggcctggag 9360

atggagacac gcgaacaagt gggcgacgag tgcgactgct gccgccacag ctcgccggtg 9420

ctcgtcatgg atcgcgagaa gatgatgtcg tcgctgcgca tgatccccag catcgtgccc 9480

ggccagcggg agatctgcct tcgcgagcgc ggctgggccg tcctcgagac ggacgcgcgc 9540

ggacactgcg agcccggcgt cctgaggctg gcgctcgccg gcctgcggct gttcgcagga 9600

tgcctgcgct ccgtcgtggg gcggcgcgag ctgtcgctgt tctgctacgg catcgctcct 9660

aagttcggcg gagagttcga ggacgcgccg cgccccatgg agatcgacgg ttagttgttt 9720

ttatccctgt acatacgccg caaactgaaa ctttagggca ccgcgtaata gtgcacgaac 9780

gcccagtgga ccgcctccgc agccatggaa aacaacgacg gtaacgaacg caacaacgaa 9840

cacccgcacg ttcgagaatt caaggaggcg tctctgtacg gttttctggt ggcggccgcg 9900

gacgtgaccg tcgaggacgt gcgccggtac cttcagttcg gcgcggacgt gaactacaag 9960

ggcgcgtacc tgtgcacgcc gctgcacgcg tacctgcagt ccggctgcga aaagcgccta 10020

gacgtcgtgg acgcgctgct ggacgccggc gcagacatca acgccaagga gatctgcggg 10080

ctcacgcccg tgcacctgta cgcgagctac gcggatgtgg acgtagagtt catgcgcggg 10140

ctcatcgagc gcggcgcgag cgtgtgcggc gagagctcgg ccacgggctg cctgtactcg 10200

tacctgtaca cacacagcgt ggacggcggc gcgcgcctgg acgtggtcga gctgctcgtg 10260

caggcgggcg cggacgtgaa cgtccgcggc gaggcgcgca agacgccgct gcacgtgcac 10320

tgcgcaagct tcgaggtgga ttcggacatc gtggagctgc tgctgcgcgc gggcgcggac 10380

cccgaggcgc tcgacgaaca cgggctcacg cccgcggacg tgctcgtgaa gtccgtgggc 10440

gccaacgtgg cgacgctgcg gctcttcctc gacgcgggcg tgagcatggc cacgtcgcgc 10500

gacgtgcgcg gacgcacgcc gctgcaccac cacgcggact cattccgggc gagtgcaggc 10560

atcgtgcgcg aactgctcgc cgccggctgc gacgcggcgg ccgccgacga cctcggaaac 10620

acgcccctgc acagcctcgc caccttctgc tcgtgccggc gctcggtgct cgaccagctc 10680

atcgccggcg gcgcggacat caacgcccgc aaccactacg gccacacctg cctgtactac 10740

gcgtccatct acaacccctc cgtttgctcg cggctcatcg ccgcgggtgc ggacgtgacc 10800

gcgcgcacgc cggacggacg cacgccgctc tcgggcatga tcatgcgcaa gcacacgcgc 10860

gccgtgcgcg ccgccctggc gacgcggcct cccgcggacg ccgtcgccgc gtcgctggac 10920

gtcgcggtac agcccgagcc cacggacgcc actcgcgcgt gcgtgcggta cgtggtgctc 10980

tgcggcggca cgctctcggc gcgcgtgcgg tcgcgacacg cggacttcgt gcgagagtgc 11040

gaaagcgagg tggtcgtgct cagaaccacc gtggtggggc tgccctgcac ctcgctgttg 11100

gacatcgtgc gtgcggcgca gccgccgccg gtgctgctct ccccgcgcgt gcaccacgtg 11160

ctgcagaagc tgcgcgtgta cgcggacttg gtggacgcgc ggctgcgcga gatgcggcac 11220

aagaccaacc tcgtggacgc ggtgtcgcgg ctcgtgtgtc cgtgcgcgct gccgccggag 11280

gtggtgcgcg gcatcctcgt gcacgtgccg acagacaacc tgcggcacac gttgaccctc 11340

ggcgtggcgc aggcctcgcg tctccttccc tcgcataaat gaaatattat tttttgtggt 11400

agaccggtac tccccgatgg accccgccgg acaacgactg cgcgcgccag ggccgtggcg 11460

cctgaacccg ccgaccgcgt ccgcgctgga aagcgcgctg ctgcggcccg cggcgtcggc 11520

gggcgccggc cgctgcgcga acgcgcacgt ggacagccgc aacatgggcg tcggcgaggg 11580

ccgcgaggtg cccgcggacg tcgaggggct catgaccgag atccacctgc ggtacggaat 11640

gacgcgcgtc caccggaacg ttcacttcgt gcagttctgg cacggcgagc acgtgcgccg 11700

gcgccccgcg cgacacgtgt tcacggtctg gatctgcctc agcggcgagg tgcgcatcta 11760

cgcagagtgc tgccaggcgg ggcacggctt cgtgctctgc cgccagatgg cggccgggta 11820

catgttcgtg accgagccca cggactcggt cacggtctcg gtgccgcacc ggctgcgcaa 11880

ctcgcggtcg ccggtgtggc tggcggcggt cttcgccacg cggcaccgcg agccgctgcc 11940

gccgcccatg tacgccgtgc ccgggcacgt ggtgctcgcg cgcagcgcct ccatgctctg 12000

cgactgctgt ccgtcggacc cgcagcgccg caacgtgatc ttctacatgc ggctgtcggg 12060

cgcgatggtg cgcgtgatcg tgccgggcgc ggagctcgag gtcgagtgca cctcggggtt 12120

ctggccggac cacttctcca tcgacgacga gtgcgtgtgc tgcgagcgtc cgcacgtcgc 12180

gcgaaccgcg gtgtggacgc tggcggagat ttgccgcggc gccacggtgg tgctcgcgcc 12240

gccactgccc cgcgactgcg ccgcggggct gctcgcggag atccgcctgg cctcgctgcg 12300

atgggtgcgc gtgcgtgcgg tccgcagcgg cagaggaagc gtgggcccgt tcccctcggt 12360

ggtgtgggcg gcagtcttct ccgccgttcg gctcttcctg gacggaaccg tgcctgcctt 12420

cccggcgtgt gtggagaacg gacgcgcggc gtacggcatg gtgtacgtgc ccccggagga 12480

gccgcggatg gacgggctct gtgtgttccc gacgcccgcc gagccggcgg cgctcttcgt 12540

ccgcggagac caggtgctcg cggccggcgc ggccgccgcc ataatcgcgg ccgctgatag 12600

gagcgtccag gccgccaatg ggtctcctgc tgccgcagag agcgtggagc aggaccggcg 12660

cgtcgagttt gaccttgggc ctaaccctga ccccagccaa gaagcgcccg cggacgcgcc 12720

gcgtgccgat tcggacaacg acaccggctc cgagaccgag accggcgacg agagtgtgga 12780

cggcgaagat gacagcgact cctcctctta ctcggtgatg tcgtcggacg acgaaaacga 12840

cagcggcgac gaggtctggg gcgactctag cgactccggc atcgaggacg acgacggcgg 12900

tgtcgggcag gccgccgagg aagaagagga ggaagagcgc gacgtcttcg gcgcggcggc 12960

ccagatgctc ggagactgac cggtggtgaa aacataaaaa taaactgttc aacacttgta 13020

ctccgggcac caacactact atccataccc accctccctc cacacactac aatggcaaac 13080

agagaagaga ttgacgcctc cgccgtcatg gctgcctacc tcgcgagaga gtacgcggcg 13140

gctgtagaag aacagctgac gccgcgcgag cgcgatgcgc tcgaagccct tcgcgtttcc 13200

ggcgaggagg tccggtcgcc gctgctgcaa gaactctcga acgcgggcga gcaccgcgcc 13260

aaccccgaaa actcgcacat ccccgccgcc ctcgtctccg cgcttctcga agcccccacc 13320

tcccccggcc gcatggtcac tgcgattgag ctctgcgcgc agatgggccg ggtatggacg 13380

cgcggccgca agctcgtcga cttcatgcgg ctcgtgtacg tgctcctaga ccgtctgccg 13440

cccacggccg acgaggacct cagcgcctgg ctgcaggccg tcgcgcgcgt gcacggcacg 13500

cggcgccgcc tgcaccgcgt tctcggcgtc ggggccgtca tggcaggcgt cggtatgctg 13560

ctgctcggcg tgcgcgtgtt gcgacgcaca taacttttta tctcggctca aactgaaata 13620

cgacattgga ctacgaaacc tataattttg ctcacggccg cgtgagataa gataataaat 13680

aacctctgag caactaacat ggccgatgag agagaggccg acggtgcgct gttccggtac 13740

ctagagagcg aggaccgtcc ggacgtgaag cacatgcgcc ggctgctgga cgagggcgcg 13800

gacgtgaact acgcgggccc gcgcgggtac gcgccgctgc acatgctcat gcgcggcaac 13860

ccgctagacc ccgacgcggt gcgactgctg ctcaacgcgg gcgcggacgt gaacgcgaca 13920

tcgctctgcg ggttcacgcc gctgcactca tacatgtgct tcgggaccgt gactccagac 13980

acgctgcgtg cgctcatgcg ccacggcgcg agcgtcagcg acctcgagcg caacatcaac 14040

gcgctgatcg agtacttcaa ccgcgacggc tgcatgggcg gcgcggaggc ggccgtgatc 14100

gcactgctgg tggagcacgg cgcgtacgta aacgccaaag acgacctcgg acgaacgccg 14160

ctgcacatct acctgtccgg cttcttcgtg tcggtacctg tggcgctcgc gctgatcgcg 14220

ctcggcgcga acccgaacgc tacggacgcg tacgggcgca cgccactgca cgccttcctg 14280

cgctcccgcg acgtggaccc cgctgtgctg aagacgctca tcgccgcggg cgcagacccg 14340

ctcgcgcgcg acatcatccg gcgcacggcg ctgcactacc actgcgagtc cttcaagacg 14400

cgcgctagtg ttatcgagac gctggtggcc gccggctgcg accccgcgag cacagacctg 14460

ctcgacaaca cggcgctgca cagcatggcc atgggcagct cctgccgcgc ctcgctaatc 14520

cgcccgctgc tggccgcggg cgtgtctgtg aacgcgcgca acgcgcggct gcagacgcca 14580

ctgcacctcg cggccgtgtt caacccgccg gcctgcgcgc ggctgctggc cgcgggcgcg 14640

gaccccgcgc tcgcggacct ggacgagaca acgccgctgc tgagcatggt gcggcacaac 14700

tgcgcacgcg cgctgcgcac ggcgctgccc ttggcgccgg acgcgctggt ggccggcgcg 14760

gtgaaccgcg tgaacgcgcg cacgccgagc gcggccacgc gcgagtgtgt gctggcgctg 14820

gcgctgcgcg gcgcgctgga cctgctgagt gcggagagcg ttgccacaca cgcggccgca 14880

atccgcgctt gcgaggcgga ggtcgcgctg ctgcggcgca cgcgcctggg cgcgccgccg 14940

acgacgctct tcgcgctgct gacaggacga ccaaacacgc tggtttccgc aaaggcggcg 15000

cgacgcgcga tggcggacgt gtgtgtctac cgcgcggtgc tggccgcgcg tgtggagcgc 15060

gtgcgccgca agtcctcgct ggtcgagcgc ctcaccgcca tggtgtgtcc gtgcgctctg 15120

ccgccagagc tagtgacgcg catcctcgcg ctcctgaccg tggaggaact cgcttgcgca 15180

attcgcaaat aataatgaac tataactagg cttattagag gcactatttg tgcagagtcg 15240

ttagttatag ttagtgtact tacaattgga atgtcgaaga acaaagttct ggtgtgtttt 15300

gtgattattc ttacttatac attatacaca gatgcgtatt gtgttgaata tgaagaaagt 15360

gaggaagata aacaacagtg cggtagtaat ggtgggcctg cgagtttacc gcacatgctc 15420

agagaactca gggccgcgtt cggaaaggta aaaactttct tccagatgaa agaccaactg 15480

aacagtatgc tactcacaca gtcgctcctc gacgacttca aaggctacct cgggtgtcag 15540

gcactttctg agatgataca gttttacttg gaagaggtga tgccgcaggc ggaaaatcac 15600

ggaccggaca tcaaagagca cgtcaactcg ctgggagaaa aactcaaaac gctgcgtctt 15660

cgactgcgtc gctgccaccg cttcctgccg tgtgagaaca agagtaaggc cgtggagcaa 15720

gtcaaacgcg tgttcaacat gctgcaggaa cgaggtgttt acaaagccat gagcgagttc 15780

gacatattca tcaactacat agaatcgtac atgactacta aaatgtaaaa atgtatacaa 15840

cttttagtta tcgtttggat tctcgtatcg ttcatactat gtatataaaa tgtatattca 15900

catagttaca gttacagtta tattcacagt tatattttta tgctcacaag atgctatata 15960

attgaaagga aattgttcac tctctgtcag ggcgccatgg actttctagg cgccgcgctt 16020

cacgactacg ttgccgacgc gcccaaggtc tgcgccgagg aggtgcggcg gctgctggcc 16080

gcgggcgcct ctgtggagta cgcgggcgag ttcgggaaga ccgcgctgca ccagtacatg 16140

ggtcgttccg gcgcggacac cgccgtcgtg cgcgcgctgc tggacgccgg cgcgcgcgtg 16200

gacctcccgg agacctgctg cggctgcacg cccgtgcacc tctgcctcat ggcggcccag 16260

atcgacgtcg aggttctccg catgctcgtc cacgagggcc gcgtcgagga ctgcggccgc 16320

gccgagctcg cctccgcggt gctcaaggag ttcgtggtga accgcgcctt cgacgagaac 16380

gtcaccgagc gagtgatgca cgttcttgtg gccgcgggcg cggacgtgaa cgccgccagc 16440

gtggtcgacc gcacgccgct gcacgtctgc ctcacgggca tgtccacgca ccctggcacc 16500

atcgccgcgc tgctgcgctt cggcgcggac gtgaacgccg tggacctctg cggcatgtcg 16560

ccgctggcgg tgctcgtgcg ctcgcgcgcg gcgaccgcag agctggtgcg catgctgctc 16620

gacgcgggcg cagacgcaca cgctgtcgac agtcgcatgg actcgctgct gcaccagcac 16680

tttcagtccg cgcgcccgcg gccggaggtg gtgcgcgagc tcatccgcca cggctgctcg 16740

ccgcgggcgc ggaaccgaat cggcaacacg ccgctgcacg aggccgcaaa acactcctcc 16800

tgcaaacact cgctggtggg gccgctgctg gctgccggcg cgagcgtgga cgcgcgaaat 16860

aacacgggca ggacgccgct ccacttggcg gcggcgtcca acccgcgcgc gtgccgccgg 16920

ctgatcgcgc ttggggcgga cgtggttgcg cgcagttacg cgggcgtcac gccgctggcg 16980

cagctggtcg cggacaataa ctccgcgctg gtgaccgcgg cgctggacac gcagcccgag 17040

ccgcgggccg tggcagagtc gctgcaagcg accacgcccg tcggcgaaac agcgtgctcg 17100

cggctctgtg tggcgtacgt ggtggcgcgc gcgccgagcg aggtcctcgg cgagcccgag 17160

cgcttcctgc acgcggcctt cgtggcggag tgcttagcgg aggtagcggc catacacgcc 17220

gtgcgctgcg gcacacctcc ggtctcgctg ctggagatcc tggtggccgc gcgcccgccg 17280

cggagcctgc tctcgcgccg cgcgcggcgg ctggccgaga gccggacgac ggtctaccgc 17340

gcgccgctcc gtgcacgcat cgcagccatg cgccatcgct cgcgactggt ggagcgcgcg 17400

ctgcgcacgc tgcgcggctg cgtgctcccg cgcgaggtgc tggagcgagt gctgcggtgt 17460

ctgtccacac aggacctgcg gacctccgga ctggccgagt agctttttct gagataagtg 17520

aataaacatg gtggaattcg atcgcgccgc caacgccacg ccatggacgc cgccgagatg 17580

gaggagctcg acatcagcgc ggagtcggcg ctgtacgact acttcatcct gaacgcggac 17640

agagcccgcg tgggcgaggt ggtcatgctt ctcgcacagg gcgcggaaat aaactacgcg 17700

gacagcttcg acaagacgcc gctgcacctg tacttgcaca cgcgacaccc gcgctcggac 17760

gtgattctgg cgctgatgga ggcgggcgcg gtcgtggaca cgccggagcg ctgctgcggc 17820

gcgaccgcgg cgcacctgta catccttaac gcggccgagg tcgacctgtc agtgctggag 17880

gccatgctga cctggggcgt gcgccagaac gaccagcact cggagcggct gctctcgagc 17940

ttgttgcgcg agtacgtggt gacccgcgcc tactcggatc agaccgagcc gatcatggac 18000

ttgctcatcg gcatgggcgc cgacgtggac atgccggtcg gcgtgagtcg cacggcgctg 18060

cacgcctgcc ttacgggcct gaacacgaac ccgtgcatga ttcgcgcgct gcttcggcgc 18120

ggcgccagcg tgaccgcaaa agacacctac gagatgacgc cgctggcggt gctgctgaag 18180

tccgcaagcg cgacgccgga actcgtgcgc atcctcgtgg aagcaggctc cgacgtgagc 18240

gccaccgact tccgcctcaa cggcatgctg caccagcacg cgcaatccac gcgcccgcgc 18300

gcgagcgtca tgcgcgagct cgtccggctg gggtgcaacc cagctgccaa aaacatgttt 18360

gggaacacgc cgatgcacat gctagccatg gaaagctcct gccgccgctc gctgatcctc 18420

ccgctgctgg aggcagggct ttccgtgaac gaggagaacc tgcactacgg caccgtgcct 18480

ctgcacgtgg cctcagggta tgacaacaca cagggctgcc tcaagctcct ccggcaggga 18540

ggagaccctg ccgtcgtatc agccgccgga cgcacgccga tctcaaacat gctcgtcaaa 18600

cgcaaccacg tggcggtcgc cggcgcgctg tcgacgcacc cgagcgcggc agtggtcgcg 18660

caggctctcg agcaggctct cgagcacgcg ctgaacgccg ggcccagcga ggcctcgcgg 18720

ctcgccgtgg cctttgtggt ggcgcgcgcc ggcgcatccg cgctaccgga ggccgttcgc 18780

cgtctgcacg agggctttgt cgccgactgc gagcgcgaag tcgcgctgct ttctcaaacc 18840

atgctcggca caccggccgt gagcgcgctg gtcgtgctgg ttagcaagga ggtctttggc 18900

actgttatct cctcgcgtgc gctgcgcgtc gtgcgggagg tccgcgtgta cgcaaggccg 18960

ctccgcgagg cgctcataaa tctgcgccac aaatgccgct tagtttccag ccttaaaagg 19020

caggtgggac cttgctcgct gcccggcgaa ctggtggagc gcgtgctcgc gaccgtgcca 19080

ctggccgact tgcgccgctc gtgcggccgc cgcgcgcccg agtgactgcc cgcacatccc 19140

gttgctacgc gactcaactg cccgctgttt ttcttccctg ttttttctta ttagatagtt 19200

gttgcctgcc tccatgatcc tcgcacgcgc cggcgggcga cctcgcacgc ccgcggcagc 19260

cgcggccgcc gccgaggacg gcaagcacag tgatcgccgg aagcgcaaga cgcccaactg 19320

cgaagacgcc gacaactccg acgacgagct agcgcagacg ccgtgcgacc gcgagtggcc 19380

ggactgtcgc gcgagctcga tcacgagctc cgactcagtc tctctcggcg acgagatcta 19440

cctgcggtac gtggcctcgc aggtggactt cgcacagacc tgggccccgc cggtgcggct 19500

gctgcgcttc ttcgggaact tctcaaagga aactctcaac cgcatgtcgc ggcgcgggta 19560

cgtgaaccgc tcctacttcc agatggcgca cgcgcgcttc tcgcccacca acgacgacat 19620

gtaccacatg gccacgggcg ggtacggcat cgtgttccgc ttcgaccgct acgtggtcaa 19680

gtacgtcttc gagcaccgca acggcatgtc cgagatggac gcctctacgg agtacacggt 19740

gccgcggttc ctgcgcaata acctcaaggg cgacgagcgc gagttcgtgg tctgcgcgct 19800

ggccatgggg ctgaactacc ggctgggctt cctgcactcg ctgtaccggc gcgtgctgca 19860

cacgctgctg ctgctcatgc gcgtggagga aggccagcgg ccctcggtgg agatgtccaa 19920

gaagccgctg ctgcgctggt tcgaggcgcg caaggacagc gagtccttcg tgcgcctgat 19980

atcgtacttc tacccctcgg ccgtgcagag caacgtgaac ctgatcaaca acttccacca 20040

cctggtgcac ttcttcgagc acgagaagcg cgcgcggtac gtgttcgacc gcggggccgt 20100

gatcgtgttc cctctggcgc gcgggtccgc ggactcaatc tcgccggagg cggcggcggc 20160

gctgggcttc gcgccgcact cggagttcct caagttcgtg ttcctgcaga tcgcgctgct 20220

gtacctaaag atctacgagc tcccgggctg cacgaacttc ctgcacgtgg acctgaagcc 20280

cgacaacgtg ctcatctttg acagcgcgcg cgcgctcagc gtgaccgcgc ccggcgcgac 20340

tttccgcttc gaggagcccg tgcgcgcggc gctgaacgac ttcgacttcg cgcgcgtggc 20400

caccatcgag aaccgcaaga tcgcgggcag cgtccgcgtg ccgcagaact ggtactacga 20460

cttccacttc ttcgcacaca cgctgctgcg cgcgtacccg cacatcgccg cggaggaccc 20520

gggcttccac acgctgctct cggagctcac ggtctcgtgc tcgcgcggga cctgcgaccg 20580

cttccggctg cgcgtgtcct cgccgcaccc catcgagcac ctcgcgcggc tggtgcgccg 20640

cgacgtgttc tcccgctgga taaatgccgc tgcagacgcc ccagacgccg cactctcctg 20700

agcccacgcc cgcggcgcca ggctcgctgt acgacgtctt cctcgcgcgc ttcctgcgcc 20760

ggctggccgc gcgcgcggcg ccggcctcag ccgcctgcgc cgtgcgcgtg agtgcggtgc 20820

gcggccgcct gcggaactgc gagctggtgg tgctgaaccg ctgccacgcg gacgcaaaca 20880

gcgcgctcgc gctggcctcc gcggcgctgg cggaaacgct ggcggcgctc cctgccgcgg 20940

acaggctcgc cgtcgcgcgc gagctgggcg tggaccccga gcacccggag ctgacgccgg 21000

accctgcctg tgcgggcgag agcgcgctcg cgcagaacat agacatccag acgctggacc 21060

tgggcgactg cggagacccc aaaggccgcc gactgcgcgt ggcgctggtg aacagcggcc 21120

acgcggccgc aaactgtgcg ctcgcgcgcg tggcgactgc gctgacgcgc cgcgtgcccg 21180

cgagccggca cggcctcgcg gagggcggca cgccgccgtg gacgctgctg ctggcggtgg 21240

ccgcggtgac ggtgctcggc gtggtggcag tctcgctgct gcggcgcgcg ctgcgggtac 21300

gctaccaatt cgcgcggccg gccgcgctgc gcgcgtagcc gcgcaaaatg taaattgtaa 21360

cgcccaactt ttaagggtgt ggtgtggcgc catgaagttg ctcgtcggca tactagtagc 21420

cgtgtgcttg caccagtatc tgctgaacgc ggacagcaac acgaaaggat ggtccgaagt 21480

gctgaaaggc agcgagtgca agcctaggcc gattgttgtt cctgtaagcg agacgcaccc 21540

agagctgact tctcagcggt tcaacccgcc gtgtgtcacg ttgatgcgat gcggcgggtg 21600

ctgcaacgac gagagcttgg aatgcgtccc cacggaagaa gtaaacgtga cgatggaact 21660

cctgggggcg tcgggctccg gtagtaacgg gatgcaacgt ctgagcttcg tagagcataa 21720

gaaatgcgat tgtagaccac gattcacaac cacgccaccg acgaccacaa ggccgcccag 21780

aagacgccgc tagcaacttt ttatggaccg cagatccaaa caatgatgcg accaggtcat 21840

gcggaaggag gcgccacggc gcaaagtgaa aaaggaccgc ctagcagtcg agaccctccc 21900

gccacggccg cggacgcccc tctcacccgc cctccgcacc acagccgaca cagcatgcac 21960

ccctcgccgc gcaggctgct cggcgagggg tgcatgctgt gtcggctgtg gtgcggaggg 22020

cgggtgagag gagttaacga cacttgttct cttaatccga gtgagagagt gagttaaccg 22080

acaacttgtt gactcgttga cttgttatct cgccatccac tctcgctcga gcgagtgagt 22140

gctgtgttag cggtcatccg ttctctcgtt ctctctctta ctccgagtga gtgagtgtgt 22200

gagttaaccg ttaaccgtta actcgttatc tagttaactt gttctcctac tctgagcgag 22260

agagcgagtc acggttaact gttccttacc cgagcgggcg ggcgataaaa ataattatat 22320

tattcgtccg tccacgaggc gaaggaaggg cggcgagggg atgctggtct aatctactaa 22380

ggccgattac aaaaacggat gggagaccgg ggaggggtca cagctccgag cggtgcatcc 22440

gcgccagctg gccgcggcgc tggcggccgc aggccgcgcg gcggggtagc cggccccagg 22500

cgcccgcagc ccttcggccg tcgaggctgt ccgcaagcgc aaagtgaaaa aggaccgcct 22560

agcagtcgag accctcccgc cacggccgcg gacgcccctc tcacccgccc tccgcaccac 22620

agccgacaca gcatgcaccc ctcgccgcgc aggctgctcg gcgcgctcgc gctggtggcg 22680

ctgggcttcc tcctcggcgg gctcttccgc cccgcggcgc cgccgctgcc ggccgccttc 22740

ctggaggcgg gccccgtccg cgcgaacggc tccgcctcgg cgacctgcct gaccgtcggc 22800

ggcgacgggc ggcacatggc ggtggtcgcg cacggcggcg ggacgctctc gccggcgtac 22860

ccgctctccg ccggcatgca cgcgagcttc tcctccgcgc gcaagggcgc gctgctgctg 22920

cacgtcgcga ccgtgcacat ctacgacgtg cgcgagctcg ggccggagtt cgagctgacc 22980

tgcgtcgcgg tggcgggcgg ctacaacgcg gcctgggcgg ccgcgcggcc cgcggccgag 23040

tggcgccgcc agctggcgcg gatgcaccgc tcggagctgt gacccctccc cggtctccca 23100

tccgtttttg taatcggcct tagtagatta gaccagcatc ccctcgccgc ccttccttcg 23160

cctcgtggac ggacgaataa tataattatt tttatcgccc gcccgctcgg gtaaggaaca 23220

gttaaccgtg actcgctctc 23240

<210> 22

<211> 183

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

Met Ala Cys Glu Cys Ala Ser Leu Ile Leu Glu Leu Leu Arg Lys Ser

1 5 10 15

Asp Asp Lys Leu Pro Ala Lys Gln Ile Ala Lys Glu Leu Gly Ile Ser

20 25 30

Lys His Glu Ala Asn Arg Gln Leu Tyr Arg Leu Leu Asp Ser Asn Asp

35 40 45

Val Cys Cys Glu Asp Gly Asn Pro Pro Arg Trp Phe Val Glu Cys Ala

50 55 60

Pro Ala Ala Pro Thr Glu Glu Asp Glu Asn Ser Asp Thr Glu Pro Met

65 70 75 80

Glu Thr Glu Ala Gly Cys Asp Thr Leu Phe Gly Gly Asp Ile Asp Ile

85 90 95

Leu Thr Gln Ser Ala Val Met Arg Leu Lys Ser Leu Asn Pro Val Ser

100 105 110

Ala Val Asn Glu Phe Cys Met Met Thr His Arg Pro Leu Glu Phe Ser

115 120 125

Glu Thr Arg Ala Gly Gly Glu Asp His Cys Pro Arg Phe Thr Cys Thr

130 135 140

Ile Thr Ile Ser Gly Lys Val Val Thr Val Ala Asp Gly Ala Ser Lys

145 150 155 160

Lys Leu Ala Arg His Thr Ala Cys Ala Ser Ala Leu Thr Val Leu Ile

165 170 175

Asn Asn Cys Gly Ile Ser Phe

180

<210> 23

<211> 288

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

Met Ala Ser Tyr Val Ser Gly Ala Ser Ala Ser Ala Asn Thr Ala Gln

1 5 10 15

Gly Gly His Ser Gln Tyr Pro Gln Tyr Tyr Ser His Thr Arg Thr Ser

20 25 30

Arg Gly Asp Val Arg Asn Glu Ser Glu Gly Arg Phe His Thr Thr Asp

35 40 45

Asp Glu His Leu Val Leu Ser Asp Asp Cys Leu Gly Asp Gly Thr Pro

50 55 60

His Cys Gly His Ser His Asn His Ser Arg Gly Asp Gly Asp Arg His

65 70 75 80

Arg Gln Arg Ala Pro Arg Leu Tyr Glu Asp Pro Val Pro Ala Asn Ile

85 90 95

Met Val Pro Thr Leu Ser Leu Glu Gln Leu Leu Glu Glu Thr Ser Val

100 105 110

Ala Gly Gly Leu Ile Gly Gly Asn Thr Glu Arg Asp Val Glu Gln Leu

115 120 125

Leu Glu Glu Phe Ser Ala Leu Cys Pro Gly Asp Gln Ile Thr Ala Leu

130 135 140

Arg Cys Met Ala Ala Ser Phe Tyr Arg Asp Ala Leu Phe Ala Pro Tyr

145 150 155 160

Ala Cys Met His Leu Ile Ala Ser Arg Met Arg Val His Tyr Ala Arg

165 170 175

Glu Val Val His Val Ala Glu Asp Leu Ala Asp Ala Met Ser Ala Asn

180 185 190

Ser Gly Val Cys Phe Arg Arg Tyr Arg Lys Arg Val Leu Glu Asp Met

195 200 205

Leu Ala Glu Glu Met Gly Val Tyr Asn Tyr Leu Ala Arg Thr Asn Ala

210 215 220

Asp Ile Cys Glu Asp Asn Leu Leu Ser Ala Val Glu Thr Leu Leu Arg

225 230 235 240

Arg Phe Arg Arg Met Gly Cys Tyr Arg Ser Leu Cys Met Leu Lys Ile

245 250 255

Leu Ala Leu Gln His Glu Asp Leu Ala Gly Phe Ile Arg Arg Ser Ile

260 265 270

Arg Lys Thr Cys Asn Phe Ala His Ala Arg Thr His Thr Val Tyr Val

275 280 285

<210> 24

<211> 188

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Met Ala Gly Arg Ala Arg Phe Ser Pro Arg Leu His Ile Pro Ala Ala

1 5 10 15

Arg Ala Ala Leu Gly Pro His Leu His Phe Pro Arg Arg Arg Leu Val

20 25 30

Leu Arg His Cys Gly Val Arg Ala Phe Val Gly Asp Ala Ile Val Ser

35 40 45

Lys Arg Glu Met Thr Asn Pro Leu Cys Ala Gln Ala Ile Val Phe Gly

50 55 60

Asn Gly Phe Val Glu Ser Tyr Val Arg Ser Leu Asp Pro Arg Leu Leu

65 70 75 80

Gly Ala Tyr His Ala Leu Ala Arg Pro Val Cys Glu Arg Pro Leu Phe

85 90 95

Ala Val Arg Gly Trp Arg Arg Leu Phe Pro Ile Val Ala Arg Arg Leu

100 105 110

Asp Ala Val Glu Arg Arg Thr Arg Arg Val Leu Arg Ser Met Cys Arg

115 120 125

Thr Tyr Ala Thr Cys Met Ser Ala Asp Arg Ala Ala Ala Val Ala His

130 135 140

Pro Val Met His Arg Arg Trp Phe Gly His Arg Ala Thr Lys Thr Arg

145 150 155 160

Arg Ala Arg Leu Arg Arg Arg Gly Arg Asn Arg Ser Ser Lys Arg Arg

165 170 175

Ala Glu Gln Arg Lys Arg Phe Cys Asn Tyr Cys Pro

180 185

<210> 25

<211> 552

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ttagaagctg atgccgcagt tgttgatgag gacggtgagt gcgctggcgc aggcggtgtg 60

gcgcgctagc ttcttgctgg ccccgtcggc cacggtaacg acctttccgg atatcgtgat 120

ggtgcaggtg aagcgcggac agtgatcctc gccgccagca cgcgtctcgg agaactccag 180

aggtctgtgt gtcatcatgc agaactcgtt gaccgcgctg accgggttaa gacttttgag 240

gcgcatcacg gcagactgag tcaggatgtc gatgtcgccg ccgaagagcg tatcgcaccc 300

agcctcggtc tccatgggct cggtgtcgga gttttcgtcc tcctcggtgg gcgcggcggg 360

cgcgcactct acgaaccagc ggggcgggtt tccgtcctcg cagcaaacgt cgttcgagtc 420

cagcaggcgg tacagctggc ggttcgcctc gtgtttggat atgccgagct ccttcgcgat 480

ctgcttggcc ggcagcttgt cgtcggattt tctgagaagc tcgaggatca gagacgcgca 540

ctcgcaagcc at 552

<210> 26

<211> 867

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

atggcctcct acgtcagcgg cgctagcgcc agcgcgaaca ccgcccaggg cggccattct 60

cagtacccac agtactattc tcacacacgc acctcccgag gcgacgtccg caacgaaagc 120

gagggtcgct tccacaccac ggacgacgag cacttggttc tgtccgatga ctgcctcggc 180

gatggcacac cacactgcgg acacagccac aaccacagtc gcggagatgg agatcggcac 240

cgccagcgcg caccgcggct ctacgaggac ccggtgcccg cgaacatcat ggtgcccacg 300

ctcagtctag agcagctgct ggaggaaacc tcggtcgcgg ggggcctcat cggcggcaac 360

acggagaggg acgtggaaca gctcctggag gagttctccg cgctctgtcc cggggaccag 420

atcaccgcgc tgcgctgcat ggcggcctcc ttctaccgcg acgcgctgtt cgcgccgtac 480

gcctgcatgc acctcatcgc cagtcggatg cgcgtgcact acgcgcgcga ggtcgtgcac 540

gtggccgagg acctcgcgga cgcgatgtcg gcgaacagcg gcgtctgctt ccggcggtac 600

cgaaagcgcg tgctagagga catgcttgcg gaggagatgg gcgtgtacaa ttacctcgcg 660

cgcaccaacg cggacatctg cgaggacaac ctgctctcgg ccgtggagac gctgctgcgg 720

cgcttccgtc ggatgggctg ctaccgctcc ctgtgcatgc tcaagatcct cgcgctgcag 780

cacgaggacc tggctggctt catccgccgc agcataagaa aaacctgcaa cttcgcgcac 840

gcgcgcacgc acacggtcta cgtgtag 867

<210> 27

<211> 567

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ttaggggcag tagttacaaa aacgtttccg ctgctcggcg cggcgtttgg aggagcggtt 60

gcggccgcgg cggcgcagcc gcgcgcggcg cgtcttcgtg gcgcggtggc cgaaccagcg 120

ccggtgcatg accgggtgcg cgaccgcggc cgcgcgatcc gcgctcatgc aggttgcgta 180

ggtgcggcac atgctgcgca gcacgcgccg cgtgcgccgc tccacggcgt cgagccgcct 240

cgcgacgatg ggaaagagcc ggcgccagcc gcgcacggcg aagagcgggc gctcgcagac 300

cgggcgcgcg agcgcgtggt aggcgcccag cagccgcggg tccagcgagc gcacgtagct 360

ctccacgaag ccgttgccaa agacgatggc ctgcgcgcag agcgggttcg tcatctccct 420

cttggagacg atggcgtcgc ccacgaaggc gcgcacgccg cagtgccgca gcaccaggcg 480

ccgccgcggg aagtgcaggt gcgggccgag cgccgcgcgg gcggcgggga tgtgcagccg 540

cggagaaaaa cgcgcgcgtc ctgccat 567

Claims

1. The application of an attenuated strain of an infectious impetigo virus ORFV-SY17 in preparing a lung tumor therapeutic drug is characterized in that the attenuated strain of the ORFV-SY17 is prepared by deleting a virulence gene of the infectious impetigo virus ORFV-SY17 strain, the virulence gene is ORFV120, and the deletion mode is as follows: 1, and the 86 th to 476 th bases of the ORFV120 nucleotide sequence are knocked out.

2. The use of claim 1, wherein the attenuated strain of sheep infectious impetigo virus is a lyophilized formulation.

3. The use of claim 1, wherein the lung tumor treatment medicament further comprises a stabilizer, and the mass ratio of the attenuated strain of the sheep infectious impetigo virus to the stabilizer is 1:1 to 100.

4. The use according to claim 3, wherein the medicament is mixed with an injectable carrier to prepare an injectable formulation, and the medicament is administered by at least one of intratumoral injection, intramuscular injection, intraperitoneal injection or intravenous injection.

5. The use according to claim 1, wherein the medicament inhibits in situ growth of lung tumors and/or inhibits metastasis of lung tumors.

6. The use of claim 5, wherein the medicament is used in combination with at least one of anti-tumor radiation therapy, anti-tumor chemotherapy, anti-tumor hormone therapy, anti-tumor small molecule targeted therapy, anti-tumor immunotherapy.