Viruses that specifically kill tumor cells and tumor treatment drugs

Abstract

This invention discloses a virus that specifically kills tumor cells and a tumor therapeutic drug, relating to the field of biotechnology. The virus is a recombinant oncolytic virus with a foreign promoter located upstream of the virus's essential gene, replacing the natural promoter of the essential gene to drive its expression in tumor cells while remaining unexpressed in normal cells. This virus can specifically kill various tumor cells, exhibiting high killing efficiency against tumor cells while remaining safe for non-tumor cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a virus that specifically kills tumor cells and a tumor therapeutic drug. Background Technology

[0002] Conquering cancer is a global challenge. Currently, cancer treatment mainly relies on traditional chemotherapy and radiotherapy, combined with the more recently developed CAR-T and antibody therapies. However, these treatments often have unsatisfactory efficacy, significant side effects, or high potential safety risks. Developing novel, safe, and highly effective treatment options is imperative. Research indicates that developing oncolytic virus therapy for cancer holds great promise.

[0003] Oncolytic viruses are a class of tumor-killing viruses capable of replication. They can replicate in tumor cells to kill these cells and trigger cancer-specific immune responses through the lysis of tumor cell fragments, enhancing their ability to kill in situ cancer cells or attack metastatic cancer cells. Utilizing this property of oncolytic viruses to treat patients' tumors is a promising therapeutic strategy.

[0004] Oncolytic viruses are genetically engineered to selectively amplify and kill cancer cells. Various oncolytic virus targeting strategies have been extensively studied. A widely used design principle is to delete non-essential genes encoding antiviral pathways in normal cells, preventing replication. However, cancerous changes alter signaling pathways, allowing oncolytic viruses to maintain replication and killing activity within cancer cells. Currently, the WNT signaling pathway genes, such as RAS, TP53, RB1, and PTEN, are well-established. Other tumor-related genes and signaling pathways, such as those involved in key viral defense in mammalian cells mediated by interferons and cytokines, are also well-established. Cancer cells disable this pathway, providing free space for viral replication and proliferation within cancer cells. The first FDA-approved oncolytic virus drug, Imlygic (T-vec), is derived from herpes simplex virus type 1 (HSV-1). It was modified by deleting the ICP34.5 and ICP47 genes. The former prevents normal cells from shutting down protein synthesis to facilitate viral replication, while the latter inhibits antigen presentation to evade antiviral immune responses. Pexa-Vec, currently in Phase 3 clinical trials, has deleted the thymosin kinase gene, allowing it to replicate only in cells with high kinase activity, such as liver cancer.

[0005] However, current oncolytic viruses have weak killing effects on cancer cells and extremely poor broad-spectrum activity. To increase the effectiveness and broad-spectrum activity of oncolytic viruses, maintaining the integrity of the viral genome should be a better design choice.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a virus and a tumor therapeutic drug that specifically kills tumor cells. It has a high killing efficiency against tumor cells and is safe for non-tumor cells. In addition, the virus has a relatively complete genome structure and can kill multiple types of tumor cells.

[0008] This invention is implemented as follows:

[0009] In a first aspect, the present invention provides a virus that specifically kills tumor cells, the virus being a recombinant oncolytic virus having an exogenous promoter on its genome, the exogenous promoter being located upstream of an essential gene of the virus to drive the expression of the essential gene in tumor cells and not in normal cells.

[0010] The virus provided by this invention is a recombinant oncolytic virus. An exogenous promoter is introduced upstream of an essential gene in the genome. This exogenous promoter replaces the natural promoter of the essential gene and drives the expression of the downstream essential gene in tumor cells. The oncolytic virus can replicate normally and kill tumor cells through its replication specificity. When in normal cells, the exogenous promoter does not drive the expression of the essential gene, affecting the replication of the oncolytic virus, thus providing better safety to normal cells.

[0011] In addition, the original genes in the genome of the recombinant oncolytic virus provided by the present invention have not been deleted or destroyed. Therefore, the recombinant oncolytic virus provided by the present invention can replicate with a strong replication ability, kill tumor cells with a high killing rate, and has a strong killing effect with a certain broad spectrum, and can kill a variety of tumor cells.

[0012] Furthermore, in some embodiments of the present invention, the exogenous promoter is a tumor cell-specific promoter;

[0013] Furthermore, in some embodiments of the present invention, the tumor cell-specific promoter is selected from any one of the following: telomerase reverse transcriptase promoter (hTERT), human epidermal growth factor receptor-2 promoter (HER-2), E2F1 promoter, osteocalcin promoter, carcinoembryonic antigen promoter, Survivin promoter, and ceruloplasmin promoter.

[0014] Furthermore, in some embodiments of the present invention, the genome of the virus also has an enhancer located between the exogenous promoter and the essential gene to enhance the expression of the essential gene;

[0015] Furthermore, in some embodiments of the present invention, the enhancer is a CMV or SV40 enhancer.

[0016] Furthermore, in some embodiments of the present invention, the essential genes in the genome of the virus are of one or more types, and each essential gene has an upstream of the exogenous promoter and the enhancer, respectively.

[0017] In other words, when there are multiple types of essential genes, each essential gene is regulated by a set of regulatory elements consisting of its own exogenous promoters and enhancers.

[0018] Furthermore, in some embodiments of the present invention, each of the essential genes has one copy or one or more additional insertions, and each copy of the essential gene has the promoter and the enhancer upstream.

[0019] In other words, if each essential gene has multiple copies, for example, two, then this essential gene will have two repeating sequences in the genome sequence. Each sequence (i.e., each copy of the gene) has the aforementioned exogenous promoter and enhancer upstream (e.g., Figure 1 (As shown in Figure A). This increase in copy number ensures that the essential gene is fully expressed even in cancer cells with extremely low transcriptional activity due to cancer cell-specific promoters, supporting effective viral replication and thus enhancing the broad-spectrum killing effect of oncolytic viruses on tumor cells.

[0020] Furthermore, in some embodiments of the present invention, the genome of the virus also includes an immunostimulatory factor expression sequence and a viral late gene promoter that drives the expression of the immunostimulatory factor expression sequence. The viral late gene promoter is regulated by the expression product of the essential gene.

[0021] Furthermore, in some embodiments of the present invention, the immunostimulatory factor expressed by the immunostimulatory factor expression sequence is interleukin-12 (IL-12) or granulocyte-macrophage colony-stimulating factor (GMCSF).

[0022] Furthermore, in some embodiments of the present invention, the viral late gene promoter is the HSV-1 glycoprotein D promoter (gD promoter) or the adenovirus late gene E3 promoter.

[0023] The viral late gene promoter is regulated by the expression products of the essential genes, ensuring that the viral late gene promoter drives the expression of downstream immunostimulatory factors (such as...) only when the essential genes are expressed. Figure 1(As shown in Figure B). This virus only expresses the essential genes in tumor cells. Therefore, when the virus provided by this invention infects tumor cells, the essential genes are expressed, which in turn regulates the expression of downstream immunostimulatory factor sequences driven by the viral late gene promoter. The expressed immunostimulatory factors can kill tumor cells. This strategy enhances its ability to kill tumor cells. However, when infecting normal cells, the immunostimulatory factors are not expressed, thus not affecting the metabolism of normal cells and ensuring its safety for normal cells.

[0024] Furthermore, in some embodiments of the present invention, the recombinant oncolytic virus is selected from any one of herpes simplex virus, Coxsackie virus, influenza virus, vaccinia virus, measles virus, poliovirus, mumps virus, vesicular stomatitis virus, Newcastle disease virus, and adenovirus.

[0025] It should be noted that different oncolytic viruses can use different essential genes, for example:

[0026] When the recombinant oncolytic virus is herpes simplex virus, the essential genes are selected from envelope glycoprotein L, uracil DNA glycosylase, capsid protein, helicogenase subunit, DNA replication initiation binding helicase, myristic acid derivative protein, deoxyribonuclease, outer serine / threonine protein kinase, DNA packaging terminal enzyme subunit 1, outer protein UL16, DNA packaging protein UL17, capsid triple-stranded subunit 2, major capsid protein, envelope protein UL20, nucleoprotein UL24, DNA packaging protein UL25, capsid maturation protease, capsid protein, envelope glycoprotein B, single-stranded DNA binding protein, DNA polymerase catalytic subunit, nuclear exit layer protein, and DNA. Packaging protein UL32, DNA packaging protein UL33, nuclear export membrane protein, capsid protein, capsid triple subunit 1, ribonucleotide reductase subunit 1, ribonucleotide reductase subunit 2, membrane host shut-off protein, DNA polymerase processing subunit, membrane protein UL45, outer envelope protein VP13 / 14, transactivator protein VP16, outer envelope protein VP22, envelope glycoprotein N, outer envelope protein UL51, helicase-primase primer subunit, envelope glycoprotein K, ICP27, nucleoprotein UL55, nucleoprotein UL56, transcription regulator ICP4, regulatory protein ICP22, envelope glycoprotein D, and membrane protein US8A are one or more of these proteins.

[0027] When the recombinant oncolytic virus is an adenovirus, the essential genes are selected from one or more of the following: early protein 1A, early protein 1B 19K, early protein 1B 55K, capsid protein Iva2, DNA polymerase, terminal protein precursor pTP, capsid protein 52K, capsid protein precursor pIIIa, pentazoc matrix, core protein pVII, core protein precursor pX, core protein precursor pVI, hexazoc, protease, single-stranded DNA binding protein, hexamer assembly protein 100K, protein 33K, capsid protein 22K, capsid protein precursor, protein U, fibrin, regulatory protein E4 open reading frame 6 / 7, regulatory protein E4 34K, regulatory protein E4 open reading frame 4, regulatory protein E4 open reading frame 3, regulatory protein E4 open reading frame 2, and regulatory protein E4 open reading frame 1.

[0028] When the recombinant oncolytic virus is vaccinia virus, the essential genes are selected from nucleotide reductase small subunit, serine / threonine kinase, DNA-binding viral core protein, polymerase large subunit, RNA polymerase subunit, DNA polymerase, thiol oxidase, presumed DNA-binding viral nucleoprotein, DNA-binding phosphoprotein, viral core cysteine ​​protease, RNA helicase NPH-II, presumed metalloproteinase, transcription elongation factor, glutathione-like protein, RNA polymerase, presumed viral nucleoprotein, late transcription factor VLTF-1, DNA-binding viral nucleoprotein, viral capsid protein, polymerase small subunit, DNA-dependent RNA polymerase subunit rpo22, and dependent... DNA-dependent RNA polymerase subunit rpo147, serine / threonine protein phosphatase, IMV heparin-binding surface protein, DNA-dependent RNA polymerase, late transcription factor VLTF-4, DNA topoisomerase type I, mRNA occluder large subunit, viral core protein 107, viral core protein 108, uracil-DNA glycosylase, triphosphatase, early gene transcription factor VETF 70kDa small subunit, DNA-dependent RNA polymerase subunit rpo18, nucleoside triphosphate hydrolase-I, mRNA occluder small subunit, rifampicin target, late transcription factor VLTF-2, late transcription factor VLTF-3, disulfide bond type The pathway, core protein 4b precursor p4b, core protein 39kDa, DNA-dependent RNA polymerase subunit rpo19, early gene transcription factor VETF 82kDa large subunit, transcription factor VITF-3 32kDa small subunit, IMV membrane protein 128, core protein 4a precursor P4a, IMV membrane protein 131, phosphorylated IMV membrane protein, IMV membrane protein A17L, DNA helicase, viral DNA polymerase processing factor, IMV membrane protein A21L, palmitoyl protein, intermediate gene transcription factor VITF-3 45kDa large subunit, DNA-dependent RNA polymerase subunit rpo132, dependent DNA RNA polymerase rpo35, IMV protein A30L, putative ATPase, serine / threonine kinase, EEV mature protein, palmitoylated EEV membrane glycoprotein, IMV surface protein A27L, EEV membrane phosphoglycoprotein, IEV and EEV membrane glycoproteins, EEV membrane glycoprotein, disulfide bond formation pathway protein, putative viral nucleoprotein, IMV membrane protein I2L, poxvirus myristoyl protein, IMV membrane protein L1R, late 16kDa putative membrane protein, putative viral membrane protein H2R, IMV membrane protein A21L, chemokine-binding protein, epidermal growth factor-like protein, and IL-18-binding protein, or one or more of these.

[0029] When the recombinant oncolytic virus is a Coxsackievirus, the essential genes are selected from one or more of the following: protein Vpg, core protein 2A, protein 2B, RNA helicase 2C, protein 3A, protease 3C, reverse transcriptase 3D, capsid protein Vp4, and protein Vp1.

[0030] When the recombinant oncolytic virus is measles virus, the essential gene is selected from one or more of nucleoprotein N, phosphoprotein P, matrix protein M, transmembrane glycoprotein F, transmembrane glycoprotein H, and RNA-dependent RNA polymerase L;

[0031] When the recombinant oncolytic virus is mumps virus, the essential gene is selected from one or more of nucleoprotein N, phosphoprotein P, fusion protein F, and RNA polymerase L;

[0032] When the recombinant oncolytic virus is a vesicular stomatitis virus, the essential gene is selected from one or more of glycoprotein G, nucleoprotein N, phosphoprotein P, and RNA polymerase L;

[0033] When the recombinant oncolytic virus is a poliovirus, the essential genes are selected from one or more of the following: capsid protein VP1, capsid protein VP2, capsid protein VP3, cysteine ​​protease 2A, protein 2B, protein 2C, protein 3A, protein 3B, protease 3C, protein 3D, and RNA-directed RNA polymerase.

[0034] When the recombinant oncolytic virus is an influenza virus, the essential genes are selected from one or more of hemagglutinin, neuraminidase, nucleoprotein, membrane protein M1, membrane protein M2, polymerase PA, polymerase PB1-F2 and polymerase PB2.

[0035] Furthermore, in some embodiments of the present invention, when the recombinant oncolytic virus is herpes simplex virus type 1 (HSV-1), the essential gene is ICP27, and the viral late gene promoter is the glycoprotein D promoter (gD promoter). The gD promoter is regulated by the ICP27 gene product.

[0036] Furthermore, in some embodiments of the present invention, when the recombinant oncolytic virus is an adenovirus, the essential gene is E1A, and the viral late gene promoter is the late gene E3 promoter. E1A gene expression can activate the E3 gene promoter.

[0037] In a second aspect, the present invention provides a tumor treatment drug containing a virus that specifically kills tumor cells as described above;

[0038] Preferably, the drug further contains a pharmaceutically acceptable carrier.

[0039] Thirdly, the present invention provides an isolated nucleic acid molecule for preparing the aforementioned virus, the nucleic acid molecule having a core sequence for insertion into a target site of a target virus, the core sequence including a promoter, the target site being located upstream of an essential gene of the target virus, the promoter being used to drive the expression of the essential gene in tumor cells and not in normal cells.

[0040] The target virus is a wild-type oncolytic virus, such as wild-type herpes simplex virus, Coxsackie virus, influenza virus, vaccinia virus, measles virus, poliovirus, mumps virus, vesicular stomatitis virus, Newcastle disease virus, or adenovirus.

[0041] Furthermore, in some embodiments of the present invention, the promoter is a tumor cell-specific promoter.

[0042] Furthermore, in some embodiments of the present invention, the tumor cell-specific promoter is selected from any one of the following: telomerase reverse transcriptase promoter (hTERT), human epidermal growth factor receptor-2 promoter (HER-2), E2F1 promoter, osteocalcin promoter, carcinoembryonic antigen promoter, Survivin promoter, and ceruloplasmin promoter.

[0043] Furthermore, in some embodiments of the present invention, the core sequence further includes an enhancer located downstream of the promoter.

[0044] Furthermore, in some embodiments of the present invention, the enhancer is selected from CMV enhancers or SV40 enhancers.

[0045] Furthermore, in some embodiments of the present invention, the core sequence further includes an immunostimulatory factor expression sequence and a viral late gene promoter that drives the expression sequence of the immunostimulatory factor.

[0046] Preferably, the immunostimulatory factor expressed by the immunostimulatory factor expression sequence is interleukin-12 or granulocyte-macrophage colony-stimulating factor.

[0047] Preferably, when the virus is HSV-1, the essential gene is ICP27, and the viral late gene promoter is the glycoprotein D promoter (gD promoter);

[0048] Alternatively, when the virus is an adenovirus, the essential gene is E1A, and the viral late gene promoter is the adenovirus late gene E3 promoter.

[0049] Furthermore, in some embodiments of the present invention, the 3' end of the core sequence has a 3' end arm, which is homologous to the downstream sequence of the target site, and the 5' end of the core sequence has a 5' end arm, which is homologous to the upstream sequence of the target site. Attached Figure Description

[0050] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 Figure 1 shows a partial genome structure diagram of the recombinant oncolytic virus provided in an embodiment of the present invention. In the figure: A: A partial genome structure diagram of the recombinant oncolytic virus 1 of Example 1, with an additional copy of an essential gene inserted, resulting in two copies of the same essential gene. Each copy of the essential gene reading frame has a regulatory element composed of a foreign tumor cell-specific promoter and an enhancer inserted upstream; A1: A partial genome structure diagram of the recombinant oncolytic virus 1 (oHSV-BJTT) with HSV-1 as the virus, ICP27 as the essential gene, hTERT as the tumor cell-specific promoter, and CMV as the enhancer; B: A partial genome structure diagram of the recombinant oncolytic virus provided in Example 2. A partial genomic structure diagram of oncolytic virus 2. Upstream of the essential gene reading frame is a first set of regulatory elements consisting of an exogenous tumor cell-specific promoter and an enhancer; downstream of the essential gene reading frame is a second set of regulatory elements consisting of a viral late gene promoter and an immunostimulatory factor. B1: A partial genomic structure diagram of recombinant oncolytic virus 2 (oHSV-BJGMCSF) with HSV-1 as the virus, HSV-1ICP27 as the essential gene, hTERT as the tumor cell-specific promoter, CMV as the enhancer, HSV-1gD as the viral late gene promoter, and GMCSF as the immunostimulatory factor.

[0052] Figure 2 Schematic diagram of the structure of plasmid pcDNA3.1-EGFP.

[0053] Figure 3ICP27 expression in African green monkey kidney cells (Vero) (normal cells) was below detectable levels using recombinant viruses oHSV-BJTT or oHSV-BJGMCSF. Vero cells were infected with 3 MOI (virus number / cell) of oHSV-BJTT, oHSV-BJGMCSF, or HSV-1 wild-type virus KOS. One day later, cells were collected, RNA was isolated, and proteins were extracted. ICP27 mRNA was detected by reverse transcription combined with semi-quantitative PCR (A), and HSV-1 ICP27 protein was detected by Western blotting (B). Figure (i): oHSV-BJTT; Figure (ii): oHSV-BJGMCSF.

[0054] Figure 4 In cancer cells, there was little difference in ICP27 protein expression between recombinant viruses oHSV-BJTT, oHSV-BJGMCSF, and wild-type virus KOS. Cancer cells HeLa, siHA, SK-BR3, and ME-180 were infected with 3 MOI of oHSV-BJTT, oHSV-BJGMCSF, or wild-type virus KOS, respectively. One day later, cells were collected, proteins were extracted, and ICP27 protein was detected using West Point Imprinting. A: oHSV-BJTT; B: oHSV-BJGMCSF.

[0055] Figure 5 The replication kinetics of oncolytic viruses oHSV-BJTT, oHSV-BJGMCSF, and wild-type virus KOS in cancer cells are basically the same. Various cancer cells were infected with 0.1 MOI of oHSV-BJTT, HSV-BJGMCSF, or KOS. After different numbers of days, cells and culture medium were harvested, and the virus remaining in the cells was released into the culture medium through three freeze-thaw cycles. Complementary cells were infected with the virus, and the viral titer (plaque-forming units / mL, PFU / mL) was determined by the plaque assay. In Figure (i): oHSV-BJTT; in Figure (ii): oHSV-BJGMCSF. In (i) and (ii): A: Cervical cancer HeLa cells; B: Cervical squamous cell carcinoma siHa cells; C: Breast cancer SK-BR3 cells; D: Breast cancer ME-180 cells.

[0056] Figure 6Recombinant oncolytic viruses oHSV-BJTT and HSV-BJGMCSF significantly inhibited the proliferation of lung cancer, gastric cancer, liver cancer, and rectal cancer in animal tumor models. Human tumor mouse models were established. After successful modeling, oncolytic viruses were injected intratumorally every 3 days for a total of 3 times. PBS (without oncolytic virus) was used as a negative control. Tumor size was measured twice weekly after oncolytic virus injection. The experiment ended when negative control animals were euthanized. Tumor growth curves were constructed based on tumor size (in the figure, A: lung cancer; B: gastric cancer; C: liver cancer; D: rectal cancer). The relative inhibition rate (E) was calculated by comparing the tumor size of the test group with that of the negative control at the end of the experiment. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0058] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0059] Example 1

[0060] The recombinant oncolytic virus 1 provided in this embodiment is obtained by modifying wild-type herpes simplex virus type 1 (KOS), and its genome sequence has the following structural characteristics:

[0061] refer to Figure 1 In embodiment A, an additional copy of an essential gene is inserted into the genome of the recombinant oncolytic virus, resulting in two copies of the essential gene. Upstream of the reading frame of each copy of the essential gene, a regulatory element consisting of a tumor cell-specific promoter and an enhancer is inserted to replace the natural promoter of the essential gene and drive its expression in tumor cells. Downstream of the reading frame of the first copy of the essential gene is an exogenous terminator 1, and downstream of the reading frame of the second copy of the essential gene is an exogenous terminator 2. The essential gene is specifically expressed in tumor cells by the regulatory element.

[0062] Specifically, in this embodiment, the essential gene is ICP27, with an additional copy of ICP27 inserted to bring the copy number to two. The tumor cell-specific promoter is hTERT, the enhancer is the CMV enhancer, exogenous terminator 1 is SV40Poly(A), and exogenous terminator 2 is BGH Poly(A). (See reference...) Figure 1 A1.

[0063] The virus will be named oHSV-BJTT in the following text.

[0064] Example 2

[0065] The recombinant oncolytic virus 2 provided in this embodiment is obtained by modifying wild-type herpes simplex virus type 1 (KOS). The genome sequence of this recombinant oncolytic virus has the following structural characteristics:

[0066] refer to Figure 1 In embodiment B, the recombinant oncolytic virus genome has a first set of regulatory elements upstream of the reading frame of the essential gene, consisting of a tumor cell-specific promoter and an enhancer; and a second set of regulatory elements downstream of the reading frame of the essential gene, consisting of a viral late gene promoter and an immunostimulatory factor reading frame. The viral late gene promoter is regulated by the expression product of the essential gene. There is an exogenous terminator 1 downstream of the reading frame of the essential gene and an exogenous terminator 2 downstream of the immunostimulatory factor reading frame.

[0067] Specifically, in this embodiment, the essential gene is ICP27, the tumor cell-specific promoter is hTERT, the enhancer is the CMV enhancer, exogenous terminator 1 is SV40Poly(A), the viral late gene promoter is the gD promoter, the immunostimulatory factor reading frame is the GMCSF reading frame, and exogenous terminator 2 is BGHPloy(A). (Reference) Figure 1 B1.

[0068] In the following text, this recombinant oncolytic virus is named oHSV-BJGMCSF.

[0069] Example 3

[0070] This embodiment provides a method for preparing the recombinant oncolytic virus provided in Embodiment 1 or 2 above, and the operation is as follows:

[0071] (1) Preparation of cells expressing ICP27

[0072] (a) Using wild-type herpes simplex virus type 1 (KOS) DNA as a template, the ICP27 coding region was amplified by PCR and inserted into the plasmid pcDNA3.1–EGFP expressing the neomycin resistance gene (structure shown in [link]). Figure 2 The HindIII and Xba sites were replaced with EGFP. This recombinant plasmid was named the ICP27 expression plasmid, and the ICP27 gene was expressed under the drive of the CMV promoter.

[0073] (b) Treat Vero cells with different concentrations of G418, replace the medium with G418 every 3 days, and observe cell death after 10 days to determine the minimum G418 concentration required for cell death, and use this as the G418 concentration for establishing cell lines.

[0074] (c) 3.5 × 10⁶ cells were implanted into each well of a 6-well cell culture plate.5 Vero cells were cultured overnight in antibiotic-free medium, and 4 μg of the ICP27 expression plasmid obtained in step (a) was transduced per well using Lipofectamine 2000. After 24 hours, the plasmids were diluted 1:20, 1:40, and 1:60 and transduced into G418-containing medium, with the medium changed every three days. After 6-7 medium changes, clones were collected and scaled up serially from 24-well plates. The protein was then isolated, and ICP27 expression was detected using Western blotting. Cells from which clones expressing high levels of ICP27 originated were selected to obtain ICP27-expressing Vero cells, named Complementary Cells C. ICP27 .

[0075] The complementary cell C ICP27 It was deposited on April 24, 2019, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, located on Luojia Mountain in Wuchang, Wuhan, with accession number CCTCC NO.C201974.

[0076] (2) Preparation of parent virus

[0077] Using wild-type herpes simplex virus type 1 as material, a recombinant virus was obtained by replacing its ICP27 with EGFP, serving as a transitional herpesvirus, i.e., the parent virus. The specific preparation method is as follows:

[0078] (a) Artificially synthesized the first nucleic acid fragment, as shown in SEQ ID NO.1:

[0079] Its upstream to downstream (5'-3') sequence includes the following elements: ICP27 5' end sequence, CMV promoter, Kozak sequence, EGFP coding frame, BGH Poly(A) and ICP27 3' end sequence;

[0080] In SEQ ID NO.1:

[0081] Positions 1-6: Irrelevant sequence; increasing the length of the ends facilitates enzyme digestion.

[0082] Positions 7-12: Xho1 site, C / TCGAG;

[0083] Bits 13-575: ICP27 5' terminal sequence;

[0084] Bits 576-1163: CMV promoter;

[0085] Positions 1164-1174: Interval sequence;

[0086] Positions 1175-1180: Kozak sequence, increases protein expression;

[0087] Positions 1181-1900: EGFP coding frame;

[0088] Positions 1901-2145: BGH Poly(A);

[0089] Positions 2146-2667: ICP27 3' terminal sequence;

[0090] Positions 2668-2673: HindIII site A / AGCTT;

[0091] Positions 2674-2679: Irrelevant sequence; increasing the end length facilitates enzyme digestion.

[0092] (b) The first nucleic acid fragment was digested with enzymes and ligated to the HindIII and Xho1 sites of the pcDNA3.1-EGFP plasmid. The resulting recombinant plasmid was named the EGFP expression plasmid.

[0093] (c) according to 3.5×10 5 The amount of cells / well, including the complementary cells C mentioned above. ICP27 Cells were seeded into 6-well cell culture plates and cultured overnight in antibiotic-free medium.

[0094] (d) Cells were infected with wild-type KOS virus at MOIs of 0.1, 0.5, 1, and 3, respectively. One hour later, the EGFP expression plasmid (4 μg DNA / well) was transduced into the cells using Lipofectamine 2000. Four hours later, the transduction solution was replaced with complete culture medium. After all cells were infected, cells and culture medium were collected, subjected to three freeze-thaw cycles, and the supernatant was collected by centrifugation. The supernatant was diluted and used to infect the complementary C cells mentioned above. ICP27 The virus was isolated using the plaque separation method. After 4-5 days, green viral plaques were selected under a fluorescence microscope, and then the obtained viral plaques were screened 2 or 3 times until pure viral plaques were obtained. The virus was then propagated and expanded to obtain the recombinant virus EGFP replacing ICP27, which was named the parent virus HSV-EGFP.

[0095] (3) Construction of recombinant plasmids

[0096] (a) The TA cloning plasmid was modified by genetic engineering so that its multiple cloning site only includes the XhoI site, and named TA-XhoI plasmid for later use.

[0097] (b) Artificially synthesize a second nucleic acid fragment, as shown in SEQ ID NO.2:

[0098] Its upstream to downstream (5'-3') sequence includes the following elements: ICP27 5' terminal sequence (excluding the natural promoter), hTERT promoter, CMV enhancer sequence, ICP27 open reading frame, SV40Poly(A) sequence, and ICP27 3' terminal sequence; in addition, the 5' and 3' ends of the second nucleic acid fragment each contain an XhoI site, and there is a HindIII site between SV40Poly(A) and the ICP27 3' terminal sequence;

[0099] In SEQ ID NO.2:

[0100] Positions 1-6: Xho1 site;

[0101] Bits 7-517: ICP27 5' end non-coding area;

[0102] Positions 518-973: hTERT promoter;

[0103] Positions 974-1039: CMV enhancer;

[0104] Bits 1040-2578: ICP27 Open Reading Box;

[0105] Positions 2579-3050: SV40Poly(A);

[0106] Positions 3051-3056: HindIII site;

[0107] Bits 3057-3576: ICP27 3' end non-coding region;

[0108] Positions 3577-3582: Xho1 site.

[0109] (c) The second nucleic acid fragment was digested with XhoI enzyme and inserted into the XhoI site of plasmid TA-XhoI. The resulting recombinant plasmid was named TA-XhoI-hTERT-CMVminimal-ICP27 plasmid and kept for later use.

[0110] (d) Artificially synthesize a third nucleic acid fragment, as shown in SEQ ID NO.3:

[0111] Its upstream to downstream (5'-3') sequence includes the following elements: hTERT promoter plus CMV enhancer sequence, ICP27 open reading frame and BGH Poly(A) sequence; the 5' and 3' ends each contain a HindIII site.

[0112] In SEQ ID NO.3:

[0113] Positions 1-6: HindIII sites;

[0114] Positions 7-462: hTERT promoter;

[0115] Positions 463-528: CMV enhancer;

[0116] Positions 529-2067: ICP27 Open Reading Box;

[0117] Positions 2068-2304: BGH Poly(A);

[0118] Positions 2305-2310: HindIII site.

[0119] (e) Artificially synthesize a fourth nucleic acid fragment, as shown in SEQ ID NO.4:

[0120] Its upstream to downstream (5'-3') components include the following: gD promoter, Kozak sequence, GMCSF open reading frame and BGH Poly(A) sequence; the 5' and 3' ends each contain a HindIII site.

[0121] In SEQ ID NO.4:

[0122] Positions 1-6: HindIII sites;

[0123] Positions 7-439: gD promoter;

[0124] Positions 440-448: Kozak sequence;

[0125] Positions 449-883: GMCSF Open Reading Box;

[0126] 884, 1100: BGH Poly(A);

[0127] Positions 1100-1115: HindIII site.

[0128] The amino acid sequence of GMCSF (SEQ ID NO.5) is as follows:

[0129] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE.

[0130] (f) The third nucleic acid fragment was digested with HindIII and inserted into the HindIII site of the TA-XhoI-hTERT-CMVminimal-ICP27 plasmid. The resulting recombinant plasmid was named ICP27-TT.

[0131] The fourth nucleic acid fragment was digested with HindIII and inserted into the HindIII site of the TA-XhoI-hTERT-CMVminimal-ICP27 plasmid. The resulting recombinant plasmid was named ICP27-GMCSF.

[0132] (4) Construction of recombinant oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF

[0133] (a) according to 3.5×10 5 The amount of cells / well, including the complementary cells C mentioned above. ICP27 Cells were seeded into 6-well cell culture plates and cultured overnight in antibiotic-free medium.

[0134] (b) Complementary cells C were infected with the above parental virus HSV-EGFP at MOIs of 0.1, 0.5, 1, and 3, respectively. ICP27 One hour later, the plasmid ICP27-TT (4 μg DNA / well) was transduced into the cells using Lipofectamine 2000. Four hours later, the transduction solution was replaced with complete culture medium. After all cells were infected, the cells and culture medium were collected, subjected to three freeze-thaw cycles, centrifuged to collect the supernatant, diluted, and used to infect the complementary C14 cells. ICP27 .

[0135] (c) Virus isolation was performed using the plaque separation method. After 4-5 days, under a fluorescence microscope, viral plaques without green fluorescence were selected. The obtained viral plaques were then screened for 2 or 3 rounds until pure viral plaques were obtained. The virus was multiplied and amplified, and DNA from infected cells was isolated. The detection fragment was amplified by PCR using specific primers and confirmed by sequencing to obtain the recombinant oncolytic virus oHSV-BJTT.

[0136] (d) Using a method similar to (a)-(c), the plasmid ICP27-GMCSF was transduced in step (b) to obtain the recombinant oncolytic virus, which is oHSV-BJGMCSF.

[0137] These two viruses were deposited on April 24, 2019, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, located on Luojia Mountain in Wuchang, Wuhan. The accession number for oHSV-BJTT virus is CCTCC NO: V201922, and the accession number for oHSV-BJGMCSF virus is CCTCC NO: V201921.

[0138] Experimental Example 1

[0139] Detection of ICP27 expression levels in normal cells infected with recombinant oncolytic virus

[0140] Methods: Vero cells were infected with 3 MOI (virus count / cell) of wild-type HSV-1 virus KOS, oHSV-BJTT, or oHSV-BJGMCSF. One day later, cells were collected, RNA was isolated, and proteins were extracted. The expression level of ICP27 mRNA was detected by reverse transcription combined with semi-quantitative PCR, and the expression level of HSV-1 ICP27 protein was detected by Western blotting. β-actin was used as a loading control for both mRNA and protein analyses.

[0141] In KOS-infected cells, both ICP27 mRNA and protein were expressed at detectable levels. In cells infected with oHSV-BJTT or oHSV-BJGMCSF, both ICP27 mRNA and protein were below detectable levels. Figure 3 (A: mRNA; B: protein). This indicates that in normal cells, ICP27 mRNA and protein are expressed at very low levels or not at all from oHSV-BJTT or oHSV-BJGMCSF.

[0142] Experiment Example 2

[0143] Detection of GMCSF and ICP27 expression levels in cancer cells infected with recombinant oncolytic virus

[0144] Methods: Cancer cells were infected with 3 MOI of oHSV-BJTT, oHSV-BJGMCSF, or wild-type KOS virus: HeLa cervical tumor cells, siHA cervical squamous cell carcinoma cells, SK-BR3 breast cancer cells, and ME-180 breast cancer cells, respectively. Six hours later, a small amount of infected cell culture medium was collected. The GMCSF content in the cell culture medium was analyzed by ELISA. One day later, all infected cells were collected, proteins were extracted, and ICP27 protein was detected using West Point Imprinting. β-actin was used as a loading control for protein analysis.

[0145] The results showed that the GMCSF content in cell cultures infected with oHSV-BJGMCSF reached detectable levels, but the GMCSF detection was negative in cell cultures infected with oHSV-BJTT or KOS (Table 1). The levels of ICP27 protein varied among different cells infected with oHSV-BJTT and oHSV-BJGMCSF, but for each cancer cell, the ICP27 protein level did not differ significantly between oHSV-BJTT / oHSV-BJGMCSF-infected cells and KOS-infected cells. Figure 4The results showed that GMCSF could be expressed from oHSV-BJGMCSF in cancer cells, while ICP27 could be specifically expressed from oHSV-BJTT and oHSV-BJGMCSF, and the expression levels were not significantly different from those from wild-type virus.

[0146] Table 1. GMCSF content (ng / ml) in different cancer cell cultures infected with oHSV-BJ GMCSF

[0147] Cancer cell types Hela siHA BR-SK3 ME-180 oHSV-BJGMCSF 4.5 5.6 9.5 7.2 oHSV-BJTT 0 0 0 0 KOS 0 0 0 0

[0148] Experimental Example 3

[0149] Detection of the proliferation kinetics of recombinant oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF in cancer cells

[0150] Methods: Various cancer cells were infected with 0.1 MOI of oHSV-BJTT, HSV-BJGMCSF, or KOS. After different numbers of days, cells and culture medium were harvested, and the virus remaining in the cells was released into the culture medium through three freeze-thaw cycles. Complementary cells C were then infected with the virus. ICP27 Viral titers (plaque formation units / mL, PFU / mL) were determined by plaque assay on days 1, 2, and 3 post-infection.

[0151] See results Figure 5 Both oHSV-BJTT(i) and HSV-BJGMCSF(ii) proliferated to varying degrees in the four types of cancer cells tested. Within the same cancer cell line (A: HeLa cells; B: siHA cells; C: SK-BR3 cells; and D: ME-180 cells), the proliferation capacity of oHSV-BJTT or HSV-BJGMCSF was slightly lower than that of KOS, but the difference was not statistically significant. These results indicate that the genetic engineering employed essentially preserved the virus's reproductive capacity, and oHSV-BJTT and oHSV-BJGMCSF exhibited good proliferation ability in cancer cells.

[0152] Experiment Example 4

[0153] Detecting the killing ability of recombinant oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF against cancer cells.

[0154] Methods: Different cancer cells, namely HeLa cells, siHA cells, SK-BR3 cells and ME-180 cells, were infected with oHSV-BJTT, oHSV-BJGMCSF or wild-type KOS at 0.25 MOI or 0.5 MOI. Cell viability was analyzed after different days. The results are shown in Tables 2-5 (oHSV-BJTT) and Tables 6-9 (oHSV-BJGMCSF).

[0155] Table 2. Survival rate (%) of HeLa cells infected with oHSV-BJTT at different MOIs

[0156] Virus (MOI 0.25) oHSV-BJTT KOS Day 1 56±3.0 47±2.8 the next day 37±1.5 25±1.3 Day 3 15±0.8 8±0.5 Day 4 5±0.2 0 Virus (MOI 0.5) oHSV-BJTT KOS Day 1 49±3.0 36±1.9 the next day 32±1.8 19±1.4 Day 3 8±0.5 4±0.2 Day 4 0 0

[0157] Table 3. Survival rate (%) of siHA cells infected with oHSV-BJTT at different MOIs

[0158] Virus (MOI 0.25) oHSV-BJTT KOS Day 1 25±2 15±0.9 the next day 5±0.5 0 Day 3 0 0 Virus (MOI 0.5) oHSV-BJTT KOS Day 1 11±0.4 5±0.4 the next day 1±0.6 0 Day 3 0 0

[0159] Table 4. Survival rate (%) of SK-BR3 cells infected with oHSV-BJTT at different MOIs

[0160]

[0161]

[0162] Table 5. Survival rate (%) of ME-180 cells infected with oHSV-BJTT at different MOIs

[0163] Virus (MOI 0.25) oHSV-BJTT KOS Day 1 18±1 9±0.5 the next day 7.4±0.4 0 Day 3 0 0 Virus (MOI 0.5) oHSV-BJTT KOS Day 1 9±0.6 7±0.4 the next day 1±0.6 0 Day 3 0 0

[0164] As can be seen from the data in Table 2-5, oHSV-BJTT has the ability to kill different cancer cells, and its ability is close to that of KOS.

[0165] Table 6. Survival rate (%) of HeLa cells infected with oHSV-BJGMCS at different MOIs

[0166] Virus (MOI 0.25) oHSV-BJGMCS KOS Day 1 62±3.5 42±2.6 the next day 41±2.4 22±1.3 Day 3 18±1.0 7±0.4 Day 4 5±0.3 0 Virus (MOI 0.5) oHSV-BJGMCS KOS Day 1 52±3.5 33±2.0 the next day 29±1.6 19±1.1 Day 3 11±0.7 3±0.2 Day 4 0 0

[0167] Table 7. Survival rate (%) of siHA cells infected with oHSV-BJGMCS at different MOIs

[0168]

[0169]

[0170] Table 8. Survival rate (%) of SK-BR3 cells infected with oHSV-BJGMCS at different MOIs

[0171] Virus (MOI 0.25) oHSV-BJGMCS KOS Day 1 24±1.2 11±0.8 the next day 0 0 Day 3 0 0 Virus (MOI 0.5) oHSV-BJGMCS KOS Day 1 8±0.5 5±0.3 the next day 1±0.06 0 Day 3 0 0

[0172] Table 9. Survival rate (%) of ME-180 cells infected with oHSV-BJGMCS at different MOIs

[0173] Virus (MOI 0.25) oHSV-BJGMCS KOS Day 1 15±0.9 9±0.6 the next day 3±0.2 0 Day 3 0 0 Virus (MOI 0.5) oHSV-BJGMCS KOS Day 1 4±0.3 3±0.2 the next day 1±0.1 0 Day 3 0 0

[0174] Table 6-9 shows that oHSV-BJGMCSF has the ability to kill different cancer cells, and its ability is not significantly different from that of KOS. These results fully demonstrate that the genetic engineering modification used in Example 3 did not significantly alter the cell-killing ability of KOS. oHSV-BJTT and oHSV-BJGMCS have tumor cell-killing abilities comparable to wild-type oncolytic viruses, and they are effective against various tumor cells, indicating that the oHSV-BJTT and oHSV-BJGMCS oncolytic viruses provided in this invention have a certain broad spectrum of activity.

[0175] Experimental Example 5

[0176] Effects of recombinant oncolytic herpesviruses oHSV-BJTT and oHSV-BJGMCSF on normal cell viability

[0177] Methods: Vero cells or primary human corneal epithelial cells were infected with oncolytic viruses oHSV-BJTT, oHSV-BJGMCSF (2 MOI), and wild-type virus KOS (0.5 MOI). Untreated cells served as a negative control. Cell viability was measured three days after infection with oHSV-BJTT and oHSV-BJGMCSF, as well as untreated cells. Cell viability was measured two days after infection with wild-type virus KOS.

[0178] The results showed that two days after KOS infection, all Vero cells or primary human corneal epithelial cells died, but the cell viability of cells infected with oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF was basically the same as that of untreated cells (Table 10). This result indicates that oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF are safe for normal cells.

[0179] Table 10. Normal cell survival rate (%) on day 3 after HSV-BJS and day 2 after KOS infection.

[0180]

[0181] Experimental Example 6

[0182] To investigate the oncolytic activity of oncolytic viruses oHSV-BJTT and oHSV-BJGMCSF, we used mouse models of human lung cancer, gastric cancer, liver cancer, and rectal cancer to test their effects on tumor proliferation. The human lung cancer, gastric cancer, and liver cancer mouse models were established by subcutaneously inoculating BALb / c (lung and gastric cancer) or NPG (liver cancer) mice with cultured human non-small cell lung cancer A549 cells, gastric cancer NCI-N87 cells, and liver cancer SK-HEP-1 cells, respectively. When the tumors reached a certain size, they were removed, cut into small pieces, and then implanted into the corresponding mice, allowing the tumors to grow to 40-120 mm.3 Intratumoral injection of the virus was initiated. A rectal cancer model was established by subcutaneously inoculating BALb / c mice with the HCT-8 rectal adenocarcinoma cell line. Mice were 4-8 weeks old and weighed 14-16 grams at the time of tumor cell inoculation. When the tumor grew to 40-120 mm... 3 Intratumoral injection of oncolytic virus (as experimental group) was initiated. The oncolytic virus was injected intratumorally every 3 days for a total of 3 times, with each injection containing 2 × 10⁻⁶ cells / mL. 7 Infective units (suspended in 40 μl PBS) were injected at multiple sites. PBS (without oncolytic virus) was used as a negative control. Each group consisted of 8 animals (the number of animals required per group was calculated based on efficacy). Tumor size was tested twice weekly after oncolytic virus injection for a total of 17-31 days (depending on the time required for euthanasia of the negative control animals). Tumor growth curves were constructed based on tumor size (the relative tumor size at the first injection was set as 1). At the end of the experiment, tumor size was compared with the negative control, and the relative inhibition rate (%) was calculated.

[0183] Relative inhibition rate (%) = (Tumor volume of negative control group - Tumor volume of experimental group / Tumor volume of negative control group - Tumor volume of control group) × 100%. A higher relative inhibition rate indicates a better inhibitory effect.

[0184] The results showed that in various tumor models, the tumor volume after injection of oHSV-BJTT and oHSV-BJGMCSF was lower than that of the negative control, indicating that oHSV-BJTT and oHSV-BJGMCSF could significantly inhibit tumor proliferation and slow down the growth of lung cancer, gastric cancer, liver cancer, and rectal cancer. Figure 6 (AE).

[0185] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. SEQUENCE LISTING <110> Wu Zetang <120> Viruses that specifically kill tumor cells and tumor treatment drugs <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 2679 <212> DNA <213> Artificial sequence <400> 1 aaatactcg agctgcgggc gctgttgttc catcatcctg tcggcatcg caatgcgatt 60 gtgttatatc gccgtggtgg ccggggtggt gctcgtggcg cttcactacg agcaggagat 120 ccagaggcgc ctgtttgatg tatgacgtca catccaggcc ggcggaacc ggaacggcat 180 atgcaactg gaactgtcc tgtcttgggg cccaccacc cgacgcgtca tatgtaatg 240 aaaatcgttc ccccgaggcc atgtgtagcc tggatccca cgaccccgcc catgggtccc 300 aattggccgt cccgttacca agaccaaccc agccagcgta tccaccccg cccgggtccc 360 cgcggaagcg gaacggtgta tgtgatatgc taattaata catgccacgt acttatggtg 420 tctgattggt ccttgtctgt gccggaggtg gggcggggc cccgcccggg gggcggaact 480 aggaggggtt tgggagcc ggccccggca ccacggtat aaggacatcc accaccggc 540 cggtggtggt gtgcagccgt gttccaacca cggtcgttga cattgattat tgactagtta 600 ttaatagtaa tcattacgg gtcattagt tcatagccca tattagt tccgcgttac 660 attackcg gtaaatggcc cgcctggctg acccccac gaccccgcc cattgacgtc 720 aataatgacg tatgttccca tagtaacgcc atagggact ttccattgac gtcaatgggt 780 ggactattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 840 gcccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 900 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 960 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1020 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1080 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1140 ggaggtctat ataagcagag ctctctggct aactgccacc atggtgagca agggcgagga 1200 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 1260 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 1320 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 1380 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 1440 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 1500 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 1560 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 1620 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 1680 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 1740 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 1800 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 1860 cgccgggatc actctcggca tggacgagct gtacaagtaa cgggcctcga ctgtgccttc 1920 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 1980 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 2040 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 2100 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggtacaat aaaaacaaaa 2160 catttcaaac aaatcgcccc acgtgttgtc cttctttgct catggccggc ggggcgtggg 2220 tcacggcaga tggcgggggt gggcccggcg tacggcctgg gtgggcggag ggaactaacc 2280 caacgtataa atccgtcccc gctccaaggc cggtgtcata gtgcccttag gagcttcccg 2340 cccgggcgca tccccccttt tgcactatga cagcgacccc cctcaccaac ctgttcttac 2400 gggccccgga cataacccac gtggcccccc cttactgcct caacgccacc tggcaggccg 2460 aaacggccat gcacaccagc aaaacggact ccgcttgcgt ggccgtgcgg agttacctgg 2520 tccgcgcctc ctgtgagacc agcggcacaa tccactgctt tttctttgcg gtatacaagg 2580 acacccacca tacccctccg ctgattaccg agctccgcaa ctttgcggac ctggttaacc 2640 acccgccggt cctacgcgaa ctggtcgaag ctttttata 2679 <210> 2 <211> 3582 <212> DNA <213> Artificial Sequence <400> 2 ctcgaggtca tcgtaggctg cgagttgatg ctacgctttg tggccgtggg tctcatcgtc 60 ggcaccgctt tcatatcccg gggggcatgt gcgatcacat accccctgtt tctgaccatc 120 accacctggt gttttgtctc caccatcggc ctgacagagc tgtattgtat tctgcggcgg 180 ggcccggccc ccaagaacgc agacaaggcc gccgccccgg ggcgatccaa ggggctgtcg 240 ggcgtctgcg ggcgctgttg ttccatcatc ctgtcgggca tcgcaatgcg attgtgttat 300 atcgccgtgg tggccggggt ggtgctcgtg gcgcttcact acgagcagga gatccagagg 360 cgcctgtttg atgtatgacg tcacatccag gccggcggaa accggaacgg catatgcaaa 420 ctggaaactg tcctgtcttg gggcccaccc acccgacgcg tcatatgtaa atgaaaatcg 480 ttcccccgag gccatgtgta gcctggatcc caacgacatg gcccctccct cgggttaccc 540 cacagcctag gccgattcga cctctctccg ctggggccct cgctggcgtc cctgcaccct 600 gggagcgcga gcggcgcgcg ggcggggaag cgcggcccag acccccgggt ccgcccggag 660 cagctgcgct gtcggggcca ggccgggctc ccagtggatt cgcgggcaca gacgcccagg 720 accgcgctcc ccacgtggcg gagggactgg ggacccgggc acccgtcctg ccccttcacc 780 ttccagctcc gcctcctccg cgcggacccc gccccgtccc gacccctccc gggtccccgg 840 cccagccccc tccgggccct cccagcccct ccccttcctt tccgcggccc cgccctctcc 900 tcgcggcgcg agtttcaggc agcgctgcgt cctgctgcgc acgtgggaag ccctggcccc 960 ggccaccccc gcgtaggcgt gtacggtggg aggcctatat aagcagagct cgtttagtga 1020 accgtcagat cgcctggaga tggcgactga cattgatatg ctaattgacc tcggcctgga 1080 cctctccgac agcgatctgg acgaggaccc ccccgagccg gcggagagcc gccgcgacga 1140 cctggaatcg gacagcaacg gggagtgttc ctcgtcggac gaggacatgg aagaccccca 1200 cggagaggac ggaccggagc cgatactcga cgccgctcgc ccggcggtcc gcccgtctcg 1260 tccagaagac cccggcgtac ccagcaccca gacgcctcgt ccgacggagc ggcagggccc 1320 caacgatcct caaccagcgc cccacagtgt gtggtcgcgc ctcggggccc ggcgaccgtc 1380 ttgctccccc gagcggcacg ggggcaaggt ggcccgcctc caacccccac cgaccaaagc 1440 ccagcctgcc cgcggcggac gccgtgggcg tcgcaggggt cggggtcgcg gtggtcccgg 1500 ggccgccgat ggtttgtcgg acccccgccg gcgtgccccc agaaccaatc gcaacccggg 1560 gggaccccgc cccggggcgg ggtggacgga cggccccggc gccccccatg gcgaggcgtg 1620 gcgcggaagt gagcagcccg acccacccgg aggcccgcgg acacggagcg tgcgccaagc 1680 accccccccg ctaatgacgc tggcgattgc ccccccgccc gcggaccccc gcgccccggc 1740 cccggagcga aaggcgcccg ccgccgacac catcgacgcc accacgcggt tggtcctgcg 1800 ctccatctcc gagcgcgcgg cggtcgaccg catcagcgag agcttcggcc gcagcgcaca 1860 ggtcatgcac gacccctttg gggggcagcc gtttcccgcc gcgaatagcc cctgggcccc 1920 ggtgctggcg ggccaaggag ggccctttga cgccgagacc agacgggtct cctgggaaac 1980 cttggtcgcc cacggcccga gcctctatcg cacttttgcc ggcaatcctc gggccgcatc 2040 gaccgccaag gccatgcgcg actgcgtgct gcgccaagaa aatttcatcg aggcgctggc 2100 ctccgccgac gagacgctgg cgtggtgcaa gatgtgcatc caccacaacc tgccgctgcg 2160 cccccaggac cccattatcg ggacggccgc ggcggtgctg gataacctcg ccacgcgcct 2220 gcggcccttt ctccagtgct acctgaaggc gcgaggcctg tgcggcctgg acgaactgtg 2280 ttcgcggcgg cgtctggcgg acattaagga cattgcatcc ttcgtgtttg tcattctggc 2340 caggctcgcc aaccgcgtcg agcgtggcgt cgcggagatc gactacgcga cccttggtgt 2400 cggggtcgga gagaagatgc atttctacct ccccggggcc tgcatggcgg gcctgatcga 2460 aatcctagac acgcaccgcc aggagtgttc gagtcgtgtc tgcgagttga cggccagtca 2520 catcgtcgcc cccccgtacg tgcacggcaa atatttttat tgcaactccc tgttttagag 2580 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 2640 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 2700 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg 2760 aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcctgcaa 2820 gcctcgtcgt cctggccgga ccacgctatc tgtgcaaggt ccccggcccc ggacgcgcgc 2880 tccatgagca gagcgcccgc cgccgaggcg aagactcggg cggcgccctg cccgtcccac 2940 caggtcaaca ggcggtaacc ggcctcttca tcgggaatgc gcgcgacctt cagcatcgcc 3000 ggcatgtccc cctggcggac gggaagtatc cagctcgacc aagctgtttt aagcttgtac 3060 aataaaaaca aaacatttca aacaaatcgc cccacgtgtt gtccttcttt gctcatggcc 3120 ggcggggcgt gggtcacggc agatggcggg ggtgggcccg gcgtacggcc tgggtgggcg 3180 gagggaacta acccaacgta taaatccgtc cccgctccaa ggccggtgtc atagtgccct 3240 taggagcttc ccgcccgggc gcatcccccc ttttgcacta tgacagcgac ccccctcacc 3300 aacctgttct tacgggcccc ggacataacc cacgtggccc ccccttactg cctcaacgcc 3360 acctggcagg ccgaaacggc catgcacacc agcaaaacgg actccgcttg cgtggccgtg 3420 cggagttacc tggtccgcgc ctcctgtgag accagcggca caatccactg ctttttcttt 3480 gcggtataca aggacaccca ccatacccct ccgctgatta ccgagctccg caactttgcg 3540 gacctggtta accacccgcc ggtcctacgc gaactgctcg ag 3582 <210> 3 <211> 2310 <212> DNA <213> Artificial Sequence <400> 3 aagcttatgg cccctccctc gggttacccc acagcctagg ccgattcgac ctctctccgc 60 tggggccctc gctggcgtcc ctgcaccctg ggagcgcgag cggcgcgcgg gcggggaagc 120 gcggcccaga cccccgggtc cgcccggagc agctgcgctg tcggggccag gccgggctcc 180 cagtggattc gcgggcacag acgcccagga ccgcgctccc cacgtggcgg agggactggg 240 gacccgggca cccgtcctgc cccttcacct tccagctccg cctcctccgc gcggaccccg 300 ccccgtcccg acccctcccg ggtccccggc ccagccccct ccgggccctc ccagcccctc 360 cccttccttt ccgcggcccc gccctctcct cgcggcgcga gtttcaggca gcgctgcgtc 420 ctgctgcgca cgtgggaagc cctggccccg gccacccccg cggtaggcgt gtacggtggg 480 aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaat ggcgactgac 540 attgatatgc taattgacct cggcctggac ctctccgaca gcgatctgga cgaggacccc 600 cccgagccgg cggagagccg ccgcgacgac ctggaatcgg acagcaacgg ggagtgttcc 660 tcgtcggacg aggacatgga agacccccac ggagaggacg gaccggagcc gatactcgac 720 gccgctcgcc cggcggtccg cccgtctcgt ccagaagacc ccggcgtacc cagcacccag 780 acgcctcgtc cgacggagcg gcagggcccc aacgatcctc aaccagcgcc ccacagtgtg 840 tggtcgcgcc tcggggcccg gcgaccgtct tgctcccccg agcggcacgg gggcaaggtg 900 gcccgcctcc aacccccacc gaccaaagcc cagcctgccc gcggcggacg ccgtgggcgt 960 cgcaggggtc ggggtcgcgg tggtcccggg gccgccgatg gtttgtcgga cccccgccgg 1020 cgtgccccca gaaccaatcg caacccgggg ggaccccgcc ccggggcggg gtggacggac 1080 ggccccggcg ccccccatgg cgaggcgtgg cgcggaagtg agcagcccga cccacccgga 1140 ggcccgcgga cacggagcgt gcgccaagca ccccccccgc taatgacgct ggcgattgcc 1200 cccccgcccg cggacccccg cgccccggcc ccggagcgaa aggcgcccgc cgccgacacc 1260 atcgacgcca ccacgcggtt ggtcctgcgc tccatctccg agcgcgcggc ggtcgaccgc 1320 atcagcgaga gcttcggccg cagcgcacag gtcatgcacg acccctttgg ggggcagccg 1380 tttcccgccg cgaatagccc ctgggccccg gtgctggcgg gccaaggagg gccctttgac 1440 gccgagacca gacgggtctc ctgggaaacc ttggtcgccc acggcccgag cctctatcgc 1500 acttttgccg gcaatcctcg ggccgcatcg accgccaagg ccatgcgcga ctgcgtgctg 1560 cgccaagaaa atttcatcga ggcgctggcc tccgccgacg agacgctggc gtggtgcaag 1620 atgtgcatcc accacaacct gccgctgcgc ccccaggacc ccattatcgg gacggccgcg 1680 gcggtgctgg ataacctcgc cacgcgcctg cggccctttc tccagtgcta cctgaaggcg 1740 cgaggcctgt gcggcctgga cgaactgtgt tcgcggcggc gtctggcgga cattaaggac 1800 attgcatcct tcgtgtttgt cattctggcc aggctcgcca accgcgtcga gcgtggcgtc 1860 gcggagatcg actacgcgac ccttggtgtc ggggtcggag agaagatgca tttctacctc 1920 cccggggcct gcatggcggg cctgatcgaa atcctagaca cgcaccgcca ggagtgttcg 1980 agtcgtgtct gcgagttgac ggccagtcac atcgtcgccc ccccgtacgt gcacggcaaa 2040 tatttttatt gcaactccct gttttaggac tgtgccttct agttgccagc catctgttgt 2100 ttgcccctcc cccgtgcctt cttgaccct ggaaggtgcc actcccactg tccttctcta 2160 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 2220 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 2280 ggtgggctct atggcttctg aggcaagctt 2310 <210> 4 <211> 1115 <212> DNA <213> Artificial Sequence <400> 4 aagcttgaat tcgccgcggg tgggggcgtt accatcccta catggaccca gttgtcgtat 60 aattttttcc ccccccccct tctccgcatg ggtgatgtcg ggtccaaact cccgacacca 120 ccagctggca tggtataaat caccggtgcg ccccccaaac catgtccggc agggggatgg 180 ggggcgaatg cggagggcac ccaacaacac cgggctaacc aggaaatccg tggccccggc 240 ccccaacaaa gatcgcggta gcccggccgt gtgacattat cgtccatacc gaccacaccg 300 acgaatcccc taagggggag gggccatttt acgaggagga ggggtataac aaagtctgtc 360 tttaaaaagc aggggttagg gagttgttcg gtcatatgct tcagtgcgaa cgaccaacta 420 ccccgatcat cagttatccg ccgccaccat gtggctgcag agcctgctgc tcttgggcac 480 tgtggcctgc agcatctctg cacccgcccg ctcgcccagc cccagcacgc agccctggga 540 gcatgtgaat gccatccagg aggcccggcg tctcctgaac ctgagtagag acactgctgc 600 tgagatgaat gaaacagtag aagtcatctc agaaatgttt gacctccagg agccgacctg 660 cctacagacc cgcctggagc tgtacaagca gggcctgcgg ggcagcctca ccaagctcaa 720 gggccccttg accatgatgg ccagccacta caagcagcac tgccctccaa ccccggaaac 780 ttcctgtgca acccagatta tcacctttga aagtttcaaa gagaacctga aggactttct 840 gcttgtcatc ccctttgact gctgggagcc agtccaggag tgagactgtg ccttctagtt 900 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 960 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 1020 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 1080 ggcatgctgg ggatgcggtg ggctctatgg agctt 1115 <210> 5 <211> 144 <212> PRT <213> Artificial Sequence <400> 5 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140

Claims

1. A virus that specifically kills tumor cells, characterized in that, The virus is either oHSV-BJTT virus or oHSVBJGMCSF virus. The accession number of oHSV-BJTT virus is CCTCC NO: V201922, and the accession number of oHSVBJGMCSF virus is CCTCC NO: V201921.

2. A tumor treatment drug, characterized in that, It contains the virus that specifically kills tumor cells as described in claim 1.

3. The tumor treatment drug according to claim 2, wherein the drug further comprises a pharmaceutically acceptable carrier.

4. A nucleic acid molecule having the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.

4.

5. A carrier, characterized in that, It contains nucleic acid molecules with sequences such as SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.

4.

6. The carrier according to claim 5, characterized in that, It contains nucleic acid molecules with sequences as shown in SEQ ID NO.2 and SEQ ID NO.3.

Images