CN110218707B

CN110218707B - Novel oncolytic virus and preparation method and application thereof

Info

Publication number: CN110218707B
Application number: CN201910455932.8A
Authority: CN
Inventors: 徐建青; 张晓燕; 丁相卿; 陈晔; 陈添悦; 廖启彬
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date: 2019-05-29
Filing date: 2019-05-29
Publication date: 2021-10-22
Anticipated expiration: 2039-05-29
Also published as: CN110218707A

Abstract

The invention discloses a novel oncolytic virus and a preparation method and application thereof, wherein the genome of the oncolytic virus comprises gene coding sequences of anti-human CTLA4 antibody and human IL-21, and can express the anti-human CTLA4 antibody and human IL-21, so that the tumor suppression effect of immunotherapy is effectively combined with the tumor suppression effect of virus therapy, the oncolytic virus capable of efficiently expressing alpha CTLA4-Fc (ALIE) -IL21 gene is prepared, and when the oncolytic virus plays a role in tumor lysis, Regulatory T Cells (Regulatory T Cells, Treg) of a tumor microenvironment are killed by massively expressing the alpha CTLA4-Fc (ALIE) or blocking the suppression effect of the Regulatory T Cells, so that the immune response of the tumor microenvironment is up-regulated, and the anti-tumor effect is improved; meanwhile, the oncolytic virus expresses a large amount of IL21, further activates the killing function of T cells and NK cells in a tumor microenvironment, and plays a multiple anti-tumor effect. Compared with simple gene therapy or virus therapy, the killing capability of the traditional Chinese medicine composition on malignant tumors is enhanced.

Description

Novel oncolytic virus and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a novel oncolytic virus containing a modified or modified high-killing-property anti-human CTLA4 antibody and a human IL-21 gene, a preparation method of the oncolytic virus and application of the oncolytic virus in the aspect of tumor resistance.

Background

Oncolytic viruses targeting tumors have attracted considerable attention in recent decades due to the limited therapeutic efficacy of standard tumor treatment regimens in the treatment of advanced tumors and serious side effects. Oncolytic viruses are increasingly becoming the first choice for anti-tumor applications due to their ability to selectively infect and lyse tumor cells locally in the tumor. The tumor selectivity of oncolytic viruses can be the tumor tropism itself or the result of genetic modification. Oncolytic viruses act on multiple cellular pathways, thereby reducing tumor resistance and also inducing different forms of cell death. In addition, oncolytic viruses can break immune tolerance of the tumor microenvironment and induce long-term tumor specific immune responses. Oncolytic viruses can specifically transport therapeutic proteins into tumor tissue, with further replication of the virus increasing expression levels in malignant cells. Alternatively, the oncolytic virus may be used in combination with chemotherapy and radiotherapy.

In recent decades, oncolytic virus therapy has attracted considerable attention for its outstanding effects, and research on it has been advancing in a long way. Currently, adenovirus (adenovirus), herpes simplex virus-1 (HSV-1), Newcastle disease virus, etc. have been successively engineered into oncolytic viruses. In 2006, oncolytic adenovirus products (oncorine) have been used in clinical treatment in china, mainly for the treatment of nasopharyngeal carcinoma and the like. The oncolytic virus is prepared by deleting E1B-55kD region of human adenovirus 5, so that the virus can propagate in cancer cells with p53 gene mutation and kill host cells, thereby generating oncolytic therapeutic effect. However, clinical data show that the therapeutic effect of such oncolytic adenovirus based on p53 gene mutation is not very desirable. Jennerex's JX-594 is a modified vaccinia virus from U.S. biotherapeutics. In a secondary clinical trial completed in 2013, it was found that the life extension time of primary liver cancer patients after high-dose injection of virus could reach 14.1 months, while those receiving low-dose injection had a life extension of only 6.7 months. The genetically engineered herpes simplex virus Oncovex GM-CSF developed by BioVex Biotechnology corporation has passed FDA approval in 2015 10 months, and has become the first oncolytic virus product in the world. The oncoVex can selectively kill tumor cells, express and secrete GM-CSF, start the organism to generate systemic immune response, and kill residual local tumor cells and metastatic tumor cells thereof. The results of a metastatic melanoma phase II trial published by BioVex in 2009 showed that 26% of 50 patients responded to treatment and 8 more patients completely recovered health. In 2013, ann in (Amgen) published the treatment data of OncoVex, and clinical trials demonstrated that OncoVex successfully reduced tumors in advanced patients, and OncoVex was more effective than other drugs of the same class in phase III studies in over 400 patients tested. Oncolytic viruses are certainly becoming a good tool for treating tumors, but there is still a need for further development and improvement of oncolytic viruses to enhance local and systemic anti-tumor effects and reduce toxic side effects.

One of the main international strategies for enhancing the anti-tumor effect of oncolytic viruses is "gene-virus therapy", i.e. the introduction of exogenous therapeutic genes into oncolytic viruses, including apoptosis-inducing genes, genes targeting the tumor microenvironment, immune regulatory genes, etc. The strategy not only opens up a new way in the field of virus treatment research, but also provides a new vector for gene therapy of cancer, and has been widely applied to the biological treatment research of tumors.

Among immunotherapeutic strategies targeting the tumor microenvironment, Regulatory T Cells (tregs) have recently become one of the hot spots of interest in the field. The research finds that the Tregs not only inhibit abnormal immune response aiming at self-antigens, but also inhibit anti-tumor immune response, a large number of Treg cells infiltrate into tumor tissues and are associated with poor prognosis, and the removal of the Treg cells can arouse and strengthen the anti-tumor immune response. However, systemic deletion of Treg cells may result in toxic autoimmune responses. Therefore, there is a need to devise a novel immunotherapeutic strategy targeting tregs in the tumor microenvironment to evoke an effective anti-tumor immune response without an autoimmune response. Compared with the classical Treg cell (FOXPro 3)⁺T cells), effector tregs (CD 4+ CD25+ T) are the major suppressor subset of tregs in the tumor microenvironment, and can act specifically on anti-tumor effector T cells, whose surface highly expresses cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) molecules. Antibodies against CTLA4 can mediate killing of effector Treg cells thereby impairing their suppressive activity. Thus, antibodies to CTLA4 were carried in oncolytic virusesThe specific removal of Treg cells will significantly improve the tumor immunotherapy effect.

In the research of anti-tumor immunotherapy strategies, in addition to antagonizing negative regulation, positive anti-tumor immune responses can be enhanced to improve the tumor killing effect. Interleukin 21 (IL-21) is a pleiotropic immunomodulatory cytokine secreted by T helper cells (Tfh), TH17 cells, and NKT cells. IL-21 inducible CD8⁺T cells secrete INF-gamma and have cytotoxicity to further enhance the killing activity, and meanwhile, IL-21 participates in the process of regulating and controlling the development and proliferation of NK cells to further improve the ADCC activity of the NK cells. The IL-21 gene carried in the oncolytic virus is expected to enhance the anti-tumor effect.

According to the invention, the oncolytic virus vaccinia virus Tiantan strain is taken as a research mode, and the fact that the vaccinia virus Tiantan strain has a strong anti-tumor effect, and the anti-tumor effect can be remarkably enhanced by carrying a gene targeting a tumor microenvironment and an immune regulatory gene. The invention particularly explains the vaccinia virus Tiantan strain and the oncolytic function of the gene and the immunoregulation gene which carry the targeted tumor microenvironment.

Disclosure of Invention

The technical problem solved by the invention is as follows: there is an urgent need for a novel oncolytic virus that can replicate in large numbers in tumor cells and eventually destroy them, and at the same time it carries a high-killing antibody against human CTLA4, a gene targeting the tumor microenvironment, helps to eliminate regulatory T cells in the tumor microenvironment, and human immunomodulatory gene IL-21 to enhance anti-tumor effects.

In order to solve the technical problems, the invention provides a novel oncolytic virus, the genome of which comprises gene coding sequences of anti-human CTLA4 antibody and human IL-21 and can express the anti-human CTLA4 antibody and the human IL-21.

Preferably, the anti-human CTLA4 antibody gene and the human IL-21 gene contained in the genome of the oncolytic virus are α CTLA4-fc (ali ie) -IL21, and the nucleotide coding sequence thereof is shown in seq id NO:3, the amino acid sequence of the expressed alpha CTLA4-Fc (ALIE) -IL21 protein is shown as SEQ ID NO:4, respectively.

Preferably, the oncolytic virus is a recombinant vaccinia virus Tiantan strain comprising alpha CTLA4-Fc (ALIE) -IL21 target gene shown in SEQ ID NO. 3, which is named as vaccinia virus Tiantan strain rTV-alpha CTLA4-Fc (ALIE) -IL21 and has the preservation number of: CCTCC NO of V201934, the preservation date is 2019, 5 and 21 months, and the preservation address is China center for type culture Collection.

Preferably, the anti-human CTLA4 antibody is α CTLA4-fc (ali), i.e.: the Fc mutant in anti-human CTLA antibody has the A330L/I332E mutation (ALIE antibody).

Preferably, the nucleotide sequences of the alpha CTLA4-Fc (ALIE) and IL-21 can be connected by T2A cleavage peptide, P2A or IRES, two nucleic acid sequences can be simultaneously expressed in a vector, and the alpha CTLA4-Fc (ALIE) in the two nucleic acid sequences can be positioned at the 5 'end or the 3' end of the sequences; or alpha CTLA4-Fc (ALIE) and IL-21 two nucleic acid sequences are expressed on different vectors but used in combination.

Preferably, the oncolytic virus is modified to encode and express an α CTLA4-Fc (ALIE) -IL21 gene, a functional fragment or variant thereof having at least 90% sequence identity to SEQ ID NO 3, SEQ ID NO 4.

Preferably, the viral backbone is derived from a modified or modified vaccinia Virus Tiantan strain, New York strain, Copenhagen strain, canary strain, Ankara strain, adenovirus vector, adeno-associated Virus vector, herpes simplex Virus vector, varicella-zoster Virus (VZV) vector, Respiratory Syncytial Virus (RSV), Liquiritian forest Virus (Semlik forest Virus, SFV), EB Virus, cytomegalovirus, human herpes Virus type 6, variola Virus, vaccinia Virus, molluscum contagiosum Virus, orf Virus, reovirus, rotavirus, enterovirus, Seneca Virus, poliovirus, coxsackie Virus, rhinovirus, hepatitis A Virus, foot and mouth disease Virus, togavirus, Samliki forest Virus, eastern equine encephalitis Virus, Dexinella Virus, and Persian Virus, Rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley fever virus, yellow fever virus, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus, arenavirus, Lassa virus, lymphocytic choriomeningitis virus, Pichimede virus, Hunning virus, Martha virus, Hantaan virus, rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, monkey virus 5, measles virus, vesicular stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, monkey immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, rous sarcoma virus, human T cell leukemia virus type 1, Rous virus, human T cell leukemia virus, Rous virus type B virus, Rous virus type B virus, Rous virus type B virus, Rous virus type B virus, Rous virus type B virus, and Rous virus type B virus, type B virus, and Rous virus type B virus, type B virus, type B virus, type B virus, type B virus, Simian foamy virus, hepatitis B virus, hepatitis E virus, human papilloma virus or polyoma virus.

Preferably, the oncolytic virus scaffold is an intracellular maturation virus, an intracellular packaging virus, a cell-associated packaging virus or an extracellular packaging virus.

Preferably, the preparation method of the alpha CTLA4-Fc (ALIE) -IL21 recombinant vaccinia virus Tiantan strain comprises the following steps:

(1) synthesizing human alpha CTLA4-Fc (ALIE) -IL21, the gene sequence of which is shown in SEQ ID NO:3 is shown in the specification;

(2) subcloning alpha CTLA4-Fc (ALIE) -IL21 into TK region of vaccinia virus shuttle plasmid (pSC65) to construct recombinant plasmid pSC 65-alpha CTLA4-Fc (ALIE) -IL 21;

(3) by means of gene homologous recombination, pSC 65-alpha CTLA4-Fc (ALIE) -IL21 plasmid is transfected into TK143 infected by wild type vaccinia virus^-In cells, the two are subjected to homologous recombination to generate recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL 21; after screening, the TK region containing SEQID NO:3, pSC 65-alpha CTLA4-fc (ali ie) -recombinant oncolytic vaccinia virus of the coding sequence of IL 21; wherein, the alpha CTLA4-Fc (ALIE) -IL21 gene is controlled by the early/late promoter p7.5 of vaccinia virus.

Preferably, in the method for preparing the alpha CTLA4-Fc (ALIE) -IL21 recombinant vaccinia virus Tiantan strain, the specific steps of using VERO cells to react with the alpha CTLA4-Fc (ALIE) -IL21 recombinant vaccinia virus Tiantan strain comprise: when the growth density of VERO cells reaches nearly 100%, dripping alpha CTLA4-Fc (ALIE) -IL21 recombinant vaccinia virus Tiantan strains, replacing a maintenance culture medium of low-concentration fetal bovine serum, inoculating about 0.02 MOI oncolytic vaccinia virus per 10 cm culture plate, putting the cells into an incubator for culture, collecting virus liquid after the recombinant poxvirus is amplified, repeatedly freezing and thawing, and performing density gradient centrifugation purification by using a sucrose solution.

In addition, the oncolytic virus of the present invention can be applied to the preparation of an anti-tumor drug, wherein the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small-cell lung cancer and non-small-cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck squamous cell carcinoma, hodgkin's lymphoma, non-hodgkin's lymphoma or hematological cancer.

Compared with the prior art, the invention has the beneficial effects that: the invention combines the gene therapy of tumor with the oncolytic effect to prepare the oncolytic virus which can efficiently express the human gene alpha CTLA4-Fc (ALIE) -IL 21. When the oncolytic virus is administered to a tumor microenvironment, the oncolytic virus can lyse tumor cells to exert an oncolytic effect, and simultaneously can express a large amount of anti-human CTLA4 antibody alpha CTLA4-Fc (ALIE) molecules so as to kill regulatory T cells in the tumor microenvironment or block the inhibitory effect of the regulatory T cells, so that the immune response of the tumor microenvironment is up-regulated, the anti-tumor effect is improved, the oncolytic virus simultaneously expresses human IL-21 in a large amount, the killing function of NK cells in the tumor microenvironment is further improved, and multiple anti-tumor effects are exerted. The oncolytic virus significantly enhances the suppression of malignant tumors relative to gene therapy or viral therapy alone.

In addition, the invention has already completed the preclinical research of the vaccinia virus Tiantan strain oncolytic virus for treating glioma, melanoma, prostatic cancer, malignant lung cancer and the like, realizes good targeting and anti-tumor effects on tumors, has a relatively complete virus amplification and quality control system, lays a foundation for further industrialization, and has good application prospect.

Drawings

FIG. 1 shows the construction of a shuttle plasmid vector for vaccinia virus Tiantan strain (expressing mouse IL-21). FIG. 1a is a diagram showing the expression profile of vaccinia virus shuttle plasmid pSC65-mIL-21 integrated with mouse IL-21 gene; FIG. 1b is a graph showing the validation of mouse IL-21 protein expression in the culture supernatant after infection of VERO cells by the vaccinia virus Tiantan strain.

FIG. 2 shows the in vivo anti-melanoma effect of recombinant vaccinia virus of mouse IL-21. FIG. 2a is a graph of the control of tumor growth in C57BL/6J mice vaccinated with B16 melanoma by wild-type vaccinia virus Tiantan strain (TV) treatment group and mouse IL-21 recombinant vaccinia virus (rTV-mIL-21) treatment group; FIG. 2B is a graph of the effect of wild-type vaccinia virus Tiantan strain treatment and recombinant vaccinia virus treatment of mouse IL-21 on the survival of B16 melanoma-inoculated C57BL/6J mice. As can be seen from the figure, the recombinant vaccinia virus of the mouse IL-21 can obviously improve the killing effect on the mouse tumor loaded with B16 melanoma in vivo, and can greatly improve the survival rate of the mouse loaded with B16 melanoma.

FIG. 3 shows the in vivo anti-glioma effect of recombinant vaccinia virus of mouse IL-21. FIG. 3a is a graph of tumor size control in C57BL/6J mice vaccinated with GL261 neuroblastoma, using a wild-type vaccinia Temple strain treatment group and a recombinant vaccinia virus treatment group for mouse IL-21; FIG. 3b is a graph of the effect of wild-type vaccinia virus Tiantan strain treatment on the survival of C57BL/6J mice vaccinated with GL261 cell neuroblastoma versus recombinant vaccinia virus treatment of mouse IL-21. As can be seen from the figure, the recombinant vaccinia virus of mouse IL-21 can obviously improve the killing effect on GL261 glioma C57BL/6J mouse tumor in vivo, and can greatly improve the survival rate of GL261 glioma-loaded mice.

FIG. 4 shows the construction of human gene α CTLA4-Fc (ALIE) -IL21 shuttle plasmid vector and the expression of α CTLA4-Fc (ALIE) -IL21 protein. FIG. 4a is an expression profile of vaccinia virus shuttle plasmid pSC65- α CTLA4-Fc (ALIE) -IL21 incorporating the α CTLA4-Fc (ALIE) -IL21 gene; FIG. 4b shows the expression of anti-human CTLA4 antibody in the supernatant secreted by recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21 after VERO cell infection; FIG. 4c is the expression of human IL-21 following infection of VERO cells with recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL 21. As shown in the figure, the recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 anti-human CTLA4 antibody and human IL-21 cytokine were successfully expressed.

FIG. 5 shows the in vivo anti-human lung cancer effect of recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL 21. FIG. 5 shows the tumor size control effect of control group, NKT cell treatment group and NKT cell combined recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21 on B-NDG (B-NSGTM) mice inoculated with NCI-H292 malignant lung cancer cells. As shown in the figure, the NKT cell combined with the recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 can obviously improve the killing effect on NCI-H292 malignant lung cancer in vivo.

FIG. 6 shows the in vivo anti-human prostate cancer effect of recombinant vaccinia virus rTV-rTV- α CTLA4-Fc (ALIE) -IL 21. FIG. 6 shows the therapeutic effect of control, NKT cell treated and NKT cell combined recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21 treated groups on LNCaP prostate cancer-inoculated B-NDG mice. As shown in the figure, the NKT cell combined with the recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 can obviously improve the killing effect on LNCaP prostate cancer in vivo.

Preservation information: vaccinia virus Tiantan strain rTV-alpha CTLA4-Fc (ALIE) -IL21, Latin literature nameOrthopoxvirus genusAnd the culture is preserved in China center for type culture Collection in 2019, 5, 21 and the address is located in Wuhan, Wuhan university, postcode 430072, and the preservation number is as follows: CCTCC NO: V201934.

Detailed Description

The first embodiment is as follows: construction and expression verification of mouse IL-21 recombinant vaccinia virus rTV-mIL-21

1.1 construction of pSC65 vector with mouse IL-21 target Gene

Artificially synthesizing a DNA sequence of the mouse-derived IL-21, wherein the synthesized sequence is shown as SEQ ID NO. 1, the amino acid sequence is shown as SEQ ID NO. 2, and the synthesized DNA sequence is used as a template to carry out PCR amplification by adopting the following primers.

The primers for amplification were:

mouse-derived IL-21-F: GTACCAGGCCTAGTACTATGGAGAGGACCCTTGTCTG

Mouse-derived IL-21-R: AATAAGCTCGAAGTCGAC CTAGGAGAGATGCTGATG

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10 seconds, 58 ℃: annealing for 30 seconds, extending for 1 minute at 72 ℃, and reacting for 30 cycles; the loop was extended for a further 10 minutes at 72 ℃ and terminated at 25 ℃.

Recovery and cloning construction of PCR products: after completion of amplification, the desired gene was isolated on 2% agarose gel, and the vector pSC65 was linearized by digestion with Sal I (Thermo Scientific, cat. No. ER 0642), and recovered by gel cutting, and the PCR fragment and the vector digested fragment were recovered using a Sanprep column DNA gel recovery kit (Promega, cat. No. A9282). The gene recovery product was ligated with the enzyme-digested linearized vector by homologous recombination (Novozak, cat. c 112-02). The ligation products were transformed into E.coli TOP10 and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly picked and sequenced, mutation sites were corrected, and after verifying that all sequences were correct, a shuttle plasmid pSC65 (pSC 65-mIL-21) of mouse-derived IL-21 gene was successfully cloned, and the plasmid construction map is shown in FIG. 1 a.

1.2 recombination of recombinant vaccinia Virus of mouse IL-21

1. Cell preparation: will be 143TK^-Cells were plated in 6-well plates at approximately 1X 10 per well⁶And (4) respectively. The culture was carried out for about 24 hours, and when the cells adhered to the wall and spread over the entire bottom surface, the next operation was carried out.

2. Vaccinia virus incubation: the cells were infected with 0.0125/3 PFU (PFU: plaque Forming Unit, virus titer)/wild type vaccinia virus Tiantan strain of the cells, incubated at 37 ℃ for 1 hour and then removed, the supernatant aspirated off, and washed once with 1 mL of PBS and then 1 mL of complete medium was added.

3. Plasmid transfection: transfection of the shuttle plasmid pSC65-mIL-21 with mouse IL-21 into 143TK^-A cell. Culturing in an incubator at 37 ℃ for about 48 hours, wherein the specific time is determined according to the cytopathic condition.

4.2 XDMEM maintenance medium (containing 2% PS and 4% FBS) for virus plating was prepared, and 2% pre-heated low melting agarose was added followed by X-gal (final concentration of 200. mu.g/mL).

5. The supernatant in the 6-well plate was aspirated off, and 4 mL of the spotting mixture was added to the 6-well plate at 300. mu.L per well. Then carefully placing the mixture into a refrigerator at 4 ℃ to promote solidification, and transferring the mixture into a incubator at 37 ℃ to perform inverted culture after the low-melting-point agarose is solidified until clear blue spots appear.

6. Recombinant viral blue plaques were picked and 500. mu.L of complete medium was added. Repeatedly freezing and thawing at-80 deg.C for more than three times to release virus as much as possible.

7. Will be 143TK^-Cells were plated in 6-well plates at approximately 1X 10 per well⁶And (4) respectively. Culture for about 24 hours until the cells adhere to the wall and spread over the entire floor.

8. Repeatedly blow the blue spots in the EP tube to completely disperse the blue spots.

9. The complete medium was changed to the maintenance medium and then the viral fluid containing the blue spots was added and incubated at 37 ℃ in an incubator for 3-4 hours.

10. Adding screening pressure: the BrdU working concentration is 50 mug/mL, and the BrdU working concentration is placed into an incubator at 37 ℃ for incubation for about 48 hours, and spot paving is carried out according to the virus spot forming condition. The purification process needs to be performed at least 5 times.

11. Then performing small sample amplification of the recombinant vaccinia virus, and paving 143TK^-Cells were plated in six well plates, 1X 10 per well⁶And (3) cells, wherein the cells are about 100% of the bottom area of the pore plate when in use.

12. The medium in the wells was changed to 2 mL of maintenance medium before inoculation. Repeatedly blowing and beating the virus liquid containing the blue spots obtained by purification until the blue spots are scattered. Approximately 100. mu.L of virus solution was added to each well. Incubating in an incubator at 37 ℃ for about 48 hours, and collecting samples according to the virus spot forming condition.

13. Collecting a sample: the culture supernatant in the wells was carefully aspirated off 1 mL. The cells were blown down well with the remaining 1 mL of medium, harvested in EP tubes and used for subsequent genome extraction and amplification as a seed virus.

1.3 verification of expression of recombinant vaccinia Virus (rTV-mIL-21) of mouse IL-21

1. 10 cm dishes were taken and inoculated about 5X 10 in each dish⁶VERO cells, ensuring that the cell density reaches 100 percent when the vaccinia virus is inoculated on the next day;

2. before virus inoculation, the complete culture medium needs to be changed into 8 mL of maintenance medium (DMEM medium +2% FBS +1% PS) so that the cells do not grow any more and virus amplification is utilized; the virus was inoculated into cells in maintenance medium at approximately 0.02 MOI (MOI = virus PFU/cell number).

3. Culturing at 37 deg.C in 5% CO2 incubator for about 48 hr, collecting infected VERO cells from one dish, washing the cells twice with PBS, collecting infected VERO cells, and analyzing the expression of mouse-derived IL-21 by Western blotting. The primary antibodies used were Anti-IL-21 mAb (Invitrogen, cat 71003) and the secondary antibodies were HRP-labeled goat-Anti-rabbit antibodies (Zhonghuajin bridge, cat zb-2301). As a result, it was revealed that, when VERO cells were infected with vaccinia virus recombinant with mouse-derived IL-21, high expression of mouse-derived IL-21 protein could be detected by Western blotting, and that a wild-type strain infected with Tiantan vaccinia virus was expressed in a small amount (FIG. 1 b).

Example two: anti-melanoma effect of recombinant vaccinia virus of mouse IL-21

2.1 amplification and purification of recombinant vaccinia Virus of mouse IL-21

VERO cell plating: 10 cm dishes, each dish being about 5X 10⁶Ensuring that the cell density reaches 100 percent when the vaccinia virus is inoculated on the next day;

2. before virus inoculation, the complete medium was replaced with 8 mL of maintenance medium (DMEM medium +2% FBS +1% PS), and the virus was inoculated into the cells in the maintenance medium at an inoculum size of about 0.02 MOI (MOI = virus PFU/cell number). Continuously culturing in an incubator at 37 ℃ and 5% CO2 for about 48 hours, and collecting samples according to the virus spot forming condition;

3. collecting vaccinia virus: discarding 8 mL of culture medium in the dish, blowing down the rest cells by using 2 mL of maintenance culture medium, and collecting in a 15 mL centrifuge tube;

4. after being frozen and stored for 24 hours, the collected virus liquid is repeatedly frozen and thawed for 2 times, the density gradient centrifugation is carried out by 36 percent of sucrose solution, the centrifugation is carried out for 90 minutes at 16000 g and 4 ℃, the supernatant is carefully poured off, the virus precipitate in a centrifugal tube is dissolved by PBS buffer solution, and the virus precipitate is subpackaged and stored at-80 ℃ to be tested for the virus titer.

2.2 titer determination of recombinant vaccinia Virus for mouse IL-21

1．143TK^-Preparation of cells: will be 143TK^-Cells were plated in 24-well plates at approximately 2X 10 per well⁵The cell density is 100 percent of the bottom area of the 24-pore plate when the cell is used;

2. diluting the virus, diluting vaccinia virus with maintenance medium（rTV-mIL-21）Diluting the virus solution by 10 times from 1:100 to obtain a final volume of 1100 μ L;

3. the complete medium in the 24-well plate was discarded, and 500. mu.L of diluted virus solution was added to the wells to make two duplicate wells. 5% CO at 37 ℃₂Incubating in the incubator for about 48 hours, and determining the spot paving time according to the virus plaque forming condition;

4. the spot paving method comprises the following steps: preparing 8 mL of spot-paving culture medium containing 2 XDMEM culture medium, 4% FBS and 2% PS and 8 mL of low-melting-point agarose which is placed in a water bath kettle at 37 ℃ after being melted in a boiling water bath, mixing the two, and adding X-gal into the mixture to obtain the final concentration of 200 mu g/mL for later use;

5. the supernatant in the 24-well plate was aspirated off. The spotting mixture of step 4 was immediately added to a 24-well plate at 500. mu.L per well. Carefully placing the agarose gel into a refrigerator at 4 ℃ to promote solidification, transferring the agarose gel into an incubator at 37 ℃ after the agarose gel with low melting point is solidified, and performing inverted culture until clear blue spots appear;

6. and (3) counting virus plaques: firstly, observing whether the number of virus plaques is gradually decreased in a ten-fold ratio trend, then counting the number of single-digit blue plaques in two multiple wells of the seed virus, and multiplying the sum of the blue plaque values in the two wells by the reciprocal value of the corresponding dilution of the well to obtain the titer of the virus in 1 mL.

2.3 in vivo anti-melanoma Effect of recombinant vaccinia Virus of mouse IL-21

1. For C57BL/6J mice (mice were implanted subcutaneously with 7X 10 cells)⁴The length and length of the tumor of the mice were recorded daily, and the volume of the tumor was calculated by the following formula.

2. Tumor volume calculation formula: tumor volume (mm)³) = (major axis × width axis)²）/2。

3. 10 days after the mice were inoculated with melanoma B16 cells, the tumorigenic mice were randomly divided into two groups (5 mice each), a wild-type vaccinia virus Tiantan strain (TV) treatment group and a recombinant vaccinia virus of mouse IL-21 (rTV-mIL-21) treatment group. The administration mode is intratumoral infusion and one-time administration.

A: TV group: 1X 10⁷PFU per mouse;

b: rTV-mIL-21 group: 1X 10⁷PFU per mouse.

4. Tumor growth curve monitoring: after cell reinfusion, tumor size was monitored daily using a vernier caliper for 30 days. The long diameter and the wide diameter of the tumor body are measured by a vernier caliper, and the tumor volume is calculated.

5. Monitoring the survival curve of the mice: according to the ethical regulations of animal experiments, when the tumor diameter of a mouse exceeds 2 cm in any direction, the mouse is euthanized, the experimental mouse is recorded as dead, and the monitoring time is 88 days.

The results are shown in the figure, compared with the wild type vaccinia virus Tiantan strain treatment group, the recombinant vaccinia virus treatment group of mouse IL-21 can significantly control the growth of melanoma of C57BL/6J mouse, and the tumor volume of 3 mice out of 5 mice is controlled at 100 mm³In the following, even total clearance (fig. 2 a). Moreover, the recombinant vaccinia virus treatment group of mouse IL-21 significantly increased survival of melanoma-loaded mice (fig. 2 b).

Example three: recombinant vaccinia virus in vivo anti-glioma effect of mouse IL-21

1. C57BL/6 mice were implanted subcutaneously with 1X 10⁶The length and the length of the tumor of the mouse were recorded every day in the glioma GL261 cells (12525), and the volume of the tumor was calculated by the following formula.

3. 7 days after the mice were inoculated with glioma GL261 cells, the tumorigenic mice were randomly divided into three groups (5 mice per group), namely a control group, a wild-type vaccinia virus Tiantan strain (TV) treatment group and a recombinant vaccinia virus (rTV-mIL-21) treatment group of mouse IL-21. The oncolytic virus was administered by intratumoral infusion back and was taken once from the purified oncolytic virus of example two.

A: control group: the same volume of physiological saline;

b: TV group: 1X 10⁷PFU per mouse;

c: rTV-mIL-21 group: 1X 10⁷PFU per mouse.

The results are shown in the figure, compared with the wild type vaccinia virus Tiantan strain treatment group, the recombinant vaccinia virus treatment group of the mouse IL-21 can significantly control the growth of glioma in C57BL/6J mice, and the tumor volume of 4 of 5 mice is controlled to be 100 mm³In the following, even total clearance (fig. 3 a). Moreover, the recombinant vaccinia virus treatment group of mouse IL-21 significantly increased survival of glioma-bearing mice (fig. 3 b).

Example four: construction and expression verification of recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21

4.1 construction of shuttle plasmid for α CTLA4-Fc (ALIE) -IL21 Using vector pSC65

Artificially synthesizing DNA sequences of human genes alpha CTLA4-Fc (ALIE) -IL21, connecting the two genes by a T2A sequence, wherein the synthesized sequence is shown as SEQ ID NO. 3, the amino acid sequence is shown as SEQ ID NO. 4, and carrying out PCR amplification by using the synthesized DNA sequence as a template and adopting the following primers.

The primers for amplification were:

αCTLA4-Fc(ALIE)-IL21-F：GTACCAGGCCTAGTACTATGGGCTGGTCTTGCATTAT

αCTLA4-Fc(ALIE)-IL21-R：AATAAGCTCGAAGTCGACTCAGGAATCTTCACTTCCGTGTGT

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10 seconds, 58 ℃: annealing for 30 seconds, extending for 2 minutes at 72 ℃, and reacting for 30 cycles; the loop was extended for a further 10 minutes at 72 ℃ and terminated at 25 ℃.

Recovery and cloning construction of PCR products: after the amplification, the target gene was isolated on 2% agarose gel, and the pSC65 vector was digested with Sal I (Thermo Scientific, cat # ER 0642) and recovered, then the PCR fragment and the vector digested fragment were recovered using a Sanprep column DNA gel recovery kit (Promega, cat # A9282), the gene recovery product and the digested linearized vector were ligated by homologous recombination (Nozak, cat # c 112-02), and the ligation product was transformed into E.coli TOP10 and grown overnight on a ampicillin-containing plate. On day 2, single colonies were randomly picked and sequenced, the mutation sites were corrected, and after confirming that all the sequences were correct, the vector pSC65 (pSC 65- α CTLA4-Fc (ALIE) -IL 21) for the α CTLA4-Fc (ALIE) -IL21 gene was successfully cloned, and the plasmid construction map is shown in FIG. 4 a.

4.2 construction of recombinant vaccinia Virus rTV- α CTLA4-Fc (ALIE) -IL21

1. Cell preparation: will be 143TK^-Cells were plated in 6-well plates at approximately 1X 10 per well⁶And (4) respectively. Culturing for about 24 hours when the cells are attached to the wall andwhen the whole bottom surface is fully paved, the next operation is carried out.

2. Vaccinia virus incubation: the cells were infected with 0.0125/3 (PFU/cell) of wild-type vaccinia virus Tiantan, incubated at 37 ℃ for 1 hour and then removed, the supernatant aspirated, and washed once with 1 mL of PBS and 1 mL of complete medium added.

3. Plasmid transfection: the shuttle plasmid pSC 65-alpha CTLA4-Fc (ALIE) -IL21 was transfected into 143TK^-A cell. Culturing in an incubator at 37 ℃ for about 48 hours, wherein the specific time is determined according to the cytopathic condition.

6. The blue spots (containing the desired recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL 21) were picked and 500. mu.L of complete medium was added. Repeatedly freezing and thawing at-80 deg.C for more than three times to release virus as much as possible.

4.3 confirmation of expression of recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21

1. Inoculating 5X 10 cm of culture dish⁶VERO cells/dish, ensuring that the cell density reaches 100% when vaccinating vaccinia virus the next day. Before virus inoculation, the complete medium needs to be changed into 8 mL of maintenance medium (DMEM medium +2% FBS +1% PS); the virus was then added at approximately 0.02 MOI. The cells were incubated at 37 ℃ in a 5% CO2 incubator for 24 hours.

2. And (3) taking a part of the infected VERO cells, fixing and penetrating the cells through a membrane, and reacting for 20min at room temperature in a dark place. After the reaction, 800. mu.l of a washing solution was added thereto, and after mixing, 12000 g was centrifuged for 30 seconds, the mixture was washed 1 to 2 times, the supernatant was discarded, and the cells were washed twice with PBS, stained with an antibody against anti-IL-21-PE (BD Pharminge, model 560463), and analyzed by flow cytometry (BD Pharminge, model forttesa). The results showed that recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL-21 was able to express the gene of interest human IL-21 in VERO cells (FIG. 4 b).

3. Meanwhile, the remaining VERO cells were further cultured at 37 ℃ in a 5% CO2 incubator for 48 hours, and cell supernatants were collected according to the formation of viral plaques, purified by Protein G Sepharose column (GE Co., Ltd., cat. No. 28-9031-34), and the purified antibodies were dissolved in PBS buffer. As shown in figure 4c, purified anti-human CTLA4 antibodies were able to bind to Treg cells expressing CTLA 4.

Example five: anti-human lung cancer effect of recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21

5.1 amplification and purification of recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21

2. before virus inoculation, the complete culture medium needs to be changed into 8 mL of maintenance medium (DMEM medium +2% FBS +1% PS) so that the cells do not grow any more and virus amplification is utilized; the virus was inoculated into cells in maintenance medium at an inoculum size of about 0.02 MOI. Continuously culturing in an incubator at 37 ℃ and 5% CO2 for about 48 hours, and collecting samples according to the virus spot forming condition;

5.2 titer determination of recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21

2. diluting the virus, namely diluting vaccinia virus solution by using a maintenance medium, and diluting the solution by 10 times from 1:100 to a final volume of 1100 mu L;

5.3 anti-human Lung cancer Effect of recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL21

1. For B-NDG mice, 5X 10 mice are planted subcutaneously⁶The length and length of the tumor of the mouse were recorded every day in the human lung cancer cells (NCI-H292, 125. mu.L), and the tumor volume was calculated by the following formula.

According to the ethical regulations of animal experiments, when the tumor diameter of a mouse exceeds 2 cm in any direction, the mouse is euthanized, and the experimental mouse is marked as dead (about 10 days of tumor formation is expected).

3. 30 days after the mice were inoculated with tumor NCI-H292 cells, the tumorigenic mice were randomly divided into four groups (5 mice each), one untreated control group and two treatment groups including a NKT cell treatment group (NKT group) and a NKT cell-combined recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 treatment group (NKT + rTV group). The administration mode is intratumoral injection and reinfusion, and the administration is carried out once.

A: control group: the same volume of physiological saline;

b: NKT group: 5X 10⁶A plurality of NKT cells;

c: NKT + rTV group: 5X 10⁶ 1X 10 combination of NKT cells⁶Recombinant vaccinia virus rTV of PFU-αCTLA4-Fc(ALIE)-IL21；

4. Tumor growth curve monitoring: after cell reinfusion, tumor size was monitored daily using a vernier caliper for 15 days. The long diameter and the wide diameter of the tumor body are measured by a vernier caliper, and the tumor volume is calculated.

As shown in FIG. 5, the NKT cell-treated group controlled the NCI-H292 tumor growth in B-NDG mice to some extent compared to the untreated control group, but the tumor volume of the NKT cell-treated mice was observed to exceed 600 mm at day 15 after tumor cell inoculation³(ii) a The NKT cell combined recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 treatment group can remarkably control the growth of the NCI-H292 lung cancer of the B-NDG mouse and can control the tumor volume of the mouse to be 100 mm³Below, even totally eliminated.

Example six: anti-human prostate cancer effect of recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21

1. For B-NDG mice, 5X 10 mice are planted subcutaneously⁶The length and the length of the tumor of the mice were recorded daily, and the volume of the tumor was calculated by the following formula.

3. 20 days after inoculation of tumor LNCaP cells, the tumorigenic mice were randomly divided into three groups (5 mice each) of an untreated control group and two treatment groups including a NKT cell treatment group (NKT group) and a NKT cell-combined recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 treatment group (NKT + rTV group). The administration mode of the oncolytic virus is intratumoral injection and reinfusion, and the administration is carried out once for a single time.

A: control group: the same volume of physiological saline;

b: NKT group: 5X 10⁶A plurality of NKT cells;

c: NKT + rTV group:5×10⁶ 1X 10 combination of NKT cells⁶PFU recombinant vaccinia virus rTV- α CTLA4-Fc (ALIE) -IL 21;

The results are shown in FIG. 6, and the NKT-treated group can control the growth of LNCaP tumor in B-NDG mice to some extent, compared with the control group, and the tumor size of 5 mice in the NKT group is 1000 mm at 15 days after tumor cell inoculation³The following. However, it was observed that the tumor volume of NKT cell-treated mice was more than 1000 mm at day 30 after tumor cell inoculation³(ii) a The NKT cell combined recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL21 treatment group can remarkably control the growth of B-NDG mouse LNCaP tumor, and can control the tumor volume of the mouse to be 100 mm³In the following, even 5 mice were completely cleared of tumors.

In conclusion, the invention combines the gene therapy of tumor with oncolytic effect to construct a novel oncolytic Virus which comprises anti-human CTLA4 antibody and human IL-21 gene coding sequence and can express the anti-human CTLA4 antibody and human IL-21, wherein the Virus skeleton of the novel oncolytic Virus is derived from modified or modified vaccinia Virus Tiantan strain, New York strain, Copenhagen strain, canary strain, Ankara strain, adenovirus vector, adeno-associated Virus vector, herpes simplex Virus vector, varicella-zoster Virus (VZV) vector, Respiratory Syncytial Virus (Respiratory Syncytial Virus, RSV), Shengliki Forest Virus (SFV), EB Virus, cytomegalovirus, human herpesvirus type 6, variola Virus, vaccinia Virus, molluscum Virus, verruca vulgaris, orf Virus, enterovirus, rotavirus, enterovirus and seniaca Virus, Poliovirus, coxsackievirus, rhinovirus, hepatitis a virus, foot and mouth disease virus, togavirus, alphavirus, semliki forest virus, eastern equine encephalitis virus, sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis c virus, japanese encephalitis virus, st louis encephalitis virus, murray valley fever virus, yellow fever virus, west nile virus, zika virus, dengue virus, ebola virus, marburg virus, arenavirus, lassa fever virus, lymphocytic choriomeningitis virus, pichinde virus, huinin virus, marupovirus, hantavirus, rift valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, monkey virus 5, measles virus, vesicular stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza a virus, hepatitis a virus, herpes virus, mumps virus, herpes virus, foot and mouth disease virus, Influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T cell leukemia virus type 1, simian foamy virus, hepatitis B virus, hepatitis E virus, human papilloma virus or polyoma virus. Can be used for controlling human lung cancer, and various solid tumors such as prostate cancer, B cell lymphoma, T cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell cancer, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck squamous cell cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or hematological tumor diseases, has very high application value for treating tumors, simple preparation and convenient mass preparation and popularization and use.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> Shanghai city public health clinic center

<120> novel oncolytic virus and preparation method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 441

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggagagga cccttgtctg tctggtagtc atcttcttgg ggacagtggc ccataaatca 60

agcccccaag ggccagatcg cctcctgatt agacttcgtc accttattga cattgttgaa 120

cagctgaaaa tctatgaaaa tgacttggat cctgaacttc tatcagctcc acaagatgta 180

aaggggcact gtgagcatgc agcttttgcc tgttttcaga aggccaaact caagccatca 240

aaccctggaa acaataagac attcatcatt gacctcgtgg cccagctcag gaggaggctg 300

cctgccagga ggggaggaaa gaaacagaag cacatagcta aatgcccttc ctgtgattcg 360

tatgagaaaa ggacacccaa agaattccta gaaagactaa aatggctcct tcaaaagatg 420

attcatcagc atctctccta g 441

<210> 2

<211> 146

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val

1 5 10 15

Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu

20 25 30

Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp

35 40 45

Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys

50 55 60

Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser

65 70 75 80

Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu

85 90 95

Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile

100 105 110

Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu

115 120 125

Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His

130 135 140

Leu Ser

145

<210> 3

<211> 1974

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggctggt cttgcattat tctgttcctc gtggccaccg ccacagagat tgtgctgacc 60

cagtctccag gcacactgtc cctgtctcct ggcgagcgcg ccaccctgag ctgccgcgcc 120

tctcagtccg tgggctcctc ttacctcgcc tggtaccagc agaagcctgg ccaggcccct 180

agactgctga tctacggcgc cttcagcaga gccacaggca tccccgaccg gttctctggc 240

tccggcaccg acttcaccct gaccatttct agactggagc ccgaggactt cgccgtgtac 300

tactgccagc agtacggctc ttccccatgg acattcggcc agggcaccaa ggtggagatc 360

aagggcggcg gcggcggcgg ccaggtgcag ctcgtggagt ctggcggcgg cgtggtgcag 420

cctggcagat ctctgagact gtcttgcgcc gcctccggct tcaccttctc tagctacacc 480

atgcactggg tgagacaggc cccaggcaag ggccttgaat gggtgacatt cattagctac 540

gacggcaaca acaagtacta cgccgacagc gtgaagggcc ggttcacaat ttcccgcgac 600

aactctaaga acacactgta cctgcagatg aactccctca gagccgagga cacagccata 660

tactactgcg cccggacagg ctggctcggc cctttcgact actggggcca gggcacactc 720

gtgaccgtgt ctagcgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 780

gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 840

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 900

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 960

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1020

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccactc 1080

cccgaggaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1140

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1200

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1260

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1320

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1380

gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa agagggcaga 1440

ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatgag atccagtcct 1500

ggcaacatgg agaggattgt catctgtctg atggtcatct tcttggggac actggtccac 1560

aaatcaagct cccaaggtca agatcgccac atgattagaa tgcgtcaact tatagatatt 1620

gttgatcagc tgaaaaatta tgtgaatgac ttggtccctg aatttctgcc agctccagaa 1680

gatgtagaga caaactgtga gtggtcagct ttttcctgct ttcagaaggc ccaactaaag 1740

tcagcaaata caggaaacaa tgaaaggata atcaatgtat caattaaaaa gctgaagagg 1800

aaaccacctt ccacaaatgc agggagaaga cagaaacaca gactaacatg cccttcatgt 1860

gattcttatg agaaaaaacc acccaaagaa ttcctagaaa gattcaaatc acttctccaa 1920

aagatgattc atcagcatct gtcctctaga acacacggaa gtgaagattc ctga 1974

<210> 4

<211> 657

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Glu

1 5 10 15

Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

20 25 30

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser Tyr

35 40 45

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

50 55 60

Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

65 70 75 80

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp

85 90 95

Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp Thr Phe

100 105 110

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly Gly Gln

115 120 125

Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser

130 135 140

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr

145 150 155 160

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Thr

165 170 175

Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys

180 185 190

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

210 215 220

Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

245 250 255

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

260 265 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

275 280 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

290 295 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

305 310 315 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

325 330 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

340 345 350

Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys

355 360 365

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

370 375 380

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

385 390 395 400

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

405 410 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

420 425 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

435 440 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

450 455 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Gly Arg

465 470 475 480

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met

485 490 495

Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met Val

500 505 510

Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp

515 520 525

Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu

530 535 540

Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu

545 550 555 560

Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys

565 570 575

Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn

580 585 590

Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly

595 600 605

Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu

610 615 620

Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln

625 630 635 640

Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp

645 650 655

Ser

Claims

1. A novel oncolytic virus is a recombinant vaccinia virus Tiantan strain containing alpha CTLA4-Fc (ALIE) -IL21 target gene shown in SEQ ID NO. 3, which is named as vaccinia virus Tiantan strain rTV-alpha CTLA4-Fc (ALIE) -IL21, and the preservation number is as follows: CCTCC NO of V201934, the preservation date is 2019, 5 and 21 months, and the preservation address is China center for type culture Collection.

2. Use of the novel oncolytic virus of claim 1 for the preparation of an anti-tumor medicament, wherein the tumor is selected from the group consisting of prostate cancer and lung cancer.

3. The method of preparing the α CTLA4-fc (ali ie) -IL21 recombinant vaccinia virus tiantan strain of claim 1, comprising the steps of:

(2) subcloning alpha CTLA4-Fc (ALIE) -IL21 into TK region of vaccinia virus shuttle plasmid pSC65 to construct recombinant plasmid pSC 65-alpha CTLA4-Fc (ALIE) -IL 21;

(3) by means of gene homologous recombination, pSC 65-alpha CTLA4-Fc (ALIE) -IL21 plasmid is transfected into TK143 infected by wild type vaccinia virus^-In cells, the two are subjected to homologous recombination to generate recombinant vaccinia virus rTV-alpha CTLA4-Fc (ALIE) -IL 21; after screening, the TK region containing the sequence SEQID NO:3 sequence of a recombinant oncolytic vaccinia virus; wherein, the alpha CTLA4-Fc (ALIE) -IL21 gene is controlled by the early/late promoter p7.5 of vaccinia virus;

(4) amplifying the obtained recombinant oncolytic vaccinia virus.

4. The method for preparing α CTLA4-Fc (ALIE) -IL21 recombinant vaccinia virus Tiantan strain as claimed in claim 3, wherein the step (4) of amplifying the recombinant oncolytic vaccinia virus comprises the following steps: when the growth density of VERO cells reaches 100%, dripping alpha CTLA4-Fc (ALIE) -IL21 recombinant vaccinia virus Tiantan strains, replacing a maintenance culture medium of low-concentration fetal bovine serum, inoculating 0.02 MOI oncolytic vaccinia virus on each 10 cm culture plate, putting the cells into an incubator for culture, collecting virus liquid after the recombinant poxvirus is amplified, repeatedly freezing and thawing, and performing density gradient centrifugation and purification by using a sucrose solution.