CN110218707A

CN110218707A - A kind of novel oncolytic virus and its preparation method and application

Info

Publication number: CN110218707A
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: 2019-09-10
Anticipated expiration: 2039-05-29
Also published as: CN110218707B

Abstract

The invention discloses a kind of novel oncolytic viruses and its preparation method and application, it include the gene coded sequence of anti-human CTLA4 antibody and human IL-2 1 in the oncolytic virus genome, and anti-human CTLA4 antibody and human IL-2 1 can be expressed, the tumor killing effect of immunization therapy is effectively combined with the oncolytic effect of viral therapy, being prepared for one kind can high efficient expression α CTLA4-Fc (ALIE)-IL21 gene oncolytic virus, while oncolytic virus plays oncolytic effect cracking tumour cell, regulatory T cells (the Regulatory T Cells of tumor microenvironment is killed by great expression α CTLA4-Fc (ALIE), Treg), or block its depression effect, to raise the immune response of tumor microenvironment, it is promoted antitumor Effect；Meanwhile oncolytic virus great expression IL21, the killing ability of T cell and NK cell in tumor microenvironment is further activated, multiple antitumous effect is played.Its killing ability to malignant tumour is enhanced relative to simple gene therapy or virus therapy.

Description

A kind of novel oncolytic virus and its preparation method and application

Technical field

The invention belongs to field of biomedicine technology, and in particular to comprising anti-human through modification or modified High Fragmentation The novel oncolytic virus of 1 gene of CTLA4 antibody and human IL-2 and the preparation method of the oncolytic virus and in antitumor side The application in face.

Background technique

Due to the limited therapeutic effect and serious side effects in standard cancer treatment scheme late oncotherapy, target Great concern is caused in recent decades to the oncolytic virus of tumour.Oncolytic virus has benefited from its own and optionally exists Tumor by local infects and cracks the characteristic of tumour cell, more and more becomes antitumor first choice.Oncolytic virus swells Tumor selectively can be the tumour taxis of itself, be also possible to the result of gene modification.Oncolytic virus may act on multiple thin Born of the same parents' access reduces the resistance of tumour with this, can also induce various forms of cell deaths.In addition, oncolytic virus can be broken The immune tolerance of tumor microenvironment induces long-term tumour-specific immune response.Oncolytic virus can specificity transhipment treatment Property albumen enter tumor tissues, the further duplication of associated virus increases the expression in malignant cell.In addition, oncolytic Virus can be used with chemotherapy and chemotherapy combined radiotherapy.

In recent decades, oncolytic virus treatment is attracted wide attention with its effect outstanding, the correlative study to it Achieve considerable progress.Adenovirus (adenovirus), (the herpes simplex virus- of herpes simplex virus -1 at present 1, HSV-1), newcastle disease virus etc. is transformed into oncolytic virus in succession.2006, oncolytic adenovirus product (oncorine) Clinical treatment is had been used in China, is mainly used for treating nasopharyngeal carcinoma etc..The oncolytic virus is by human adenovirus type 5 E1B-55kD Area is deleted, and be bred the virus can in the cancer cell of p53 gene mutation and is killed host cell, and oncolytic therapy effect is generated. But clinical data shows that the therapeutic effect of this oncolytic adenovirus based on p53 gene mutation is not very ideal. The JX-594 of Jennerex is the modified vaccinia virus of one kind of biological therapy company, the U.S..In the second phase that 2013 complete In clinical test, Patients with Primary is found after having injected the virus of high dose, life prolongs prolonged median can Reach 14 .1 months, and the patient for receiving low dosage injection only has 6 .7 months life extended periods.BioVex biotechnology is public The genetically engineered herpes simplex virus OncoVEX GM-CSF of department's research and development has passed through the approval of FDA in October, 2015, Oncolytic virus product as the first listing in the world.OncoVex is selectively killing tumour cell simultaneously, expression secretion GM- CSF starts the immune response of body generation system, kills the tumour cell of remaining local tumor cell and its transfer.2009 BioVex announce a metastasis melanin tumor II phase test the results show that have in 50 patients 26% pair treatment have instead It answers, more there is 8 patient's complete recovery of health.Pacify into (Amgen) and disclose the treatment data of OncoVex in March, 2013, it is clinical The tumor regression of patients with terminal can successfully be allowed by proving it, and in the III phase for testing patient more than 400 is studied, OncoVex is wanted It is more more efficient than similar other drugs.Oncolytic virus is becoming the sharp weapon for the treatment of tumour really, but oncolytic virus still needs It further develops and improves, to enhance part and systemic anti-neoplastic effect, and reduce toxic side effect.

In order to enhance the anti-tumor effect of oncolytic virus, the main policies used in the world first is that " gene-virus is controlled Treat ", i.e., by Exogenous therapies channel genes oncolytic virus, the gene including inducing cell apoptosis gene, target tumor microenvironment With immunomodulatory gene etc..This strategy not only opened up a new way in viral therapy research field, also control for the gene of cancer Treatment provides new carrier, has been widely used in tumor biotherapy research.

In the Immunotherapy Strategy of target tumor microenvironment, regulatory T cells (Regulatory T Cells, Treg) one of the hot spot paid close attention in the recent period as field.It is answered the study found that Treg not only inhibits abnormal for the immune of autoantigen It answers, and inhibits anti-tumor immune response, it is related to undesirable prognosis that a large amount of Treg cellular infiltrations enter tumor tissues, removal Treg cell can arouse and strengthen anti-tumor immune response.But systematic deletion Treg cell may cause it is toxic Autoimmune response.Therefore, it is necessary to the Immunotherapy Strategy of Treg in novel targeted tumor microenvironment a kind of be designed, to call out It plays the anti-tumor immune response of validity and not will cause autoimmune response simultaneously.Compared to classical Treg cell (FOXPro3⁺T cell), effect Treg(CD4+CD25+T) it is main suppressive Treg subgroup in tumor microenvironment, it can Specificity acts on antitumor T effector cell, surface high expressing cell poison T lymphocyte antigen 4 (cytotoxic T-lymphocyte-associated protein 4, CTLA-4) molecule.It can be situated between for the antibody of CTLA4 Lethal effect Treg cell is led to weaken its inhibitory activity.Therefore, the antibody of CTLA4 is carried in oncolytic virus with specificity Treg cell is removed, immunotherapy of tumors effect will be obviously improved.

In antitumor Immunotherapy Strategy research, other than the effect of antagonism negative regulation, reinforcing can also be passed through Positive anti-tumor immune response is to improve tumor-killing effect.Interleukin 21 (Interleukin-21, IL-21) is a kind of multiple-effect Property immunomodulating cytokines are thin by t helper cell (T follicular helper cells, Tfh), TH17 cell and NKT Intracrine.IL-21 can induce CD8⁺T cell secretion INF- γ and cytotoxicity enhance lethal activity in turn, meanwhile, IL- 21 participate in the development of regulation NK cell and birth process and then the ADCC activity for promoting NK cell.IL- is carried in oncolytic virus 21 genes are also expected to enhance its antitumor action.

Using oncolytic virus vaccinia virus Tiantan strain as research mode, discovery vaccinia virus Tiantan strain itself has strong the present invention Big antitumor action, the gene and immunomodulatory gene for carrying target tumor microenvironment can significantly increase antitumous effect.This Invention will focus on the oncolytic of the gene and immunomodulatory gene that illustrate vaccinia virus Tiantan strain and its carrying target tumor microenvironment Function.

Summary of the invention

Technical problem solved by the invention is: being badly in need of a kind of novel oncolytic virus, which can be thin in tumour Massive duplication intracellular and final destroyed tumor cell, while it also carries the height of the anti-human CTLA4 of gene of target tumor microenvironment Lethal antibody, the immunomodulatory gene IL-21 for assisting to remove the regulatory T-cell and people in tumor microenvironment are anti-to enhance Tumor effect.

It is viral that in order to solve the above technical problem, the present invention provides a kind of novel oncolytics, in the oncolytic virus genome Gene coded sequence comprising anti-human CTLA4 antibody and human IL-2 1, and anti-human CTLA4 antibody and human IL-2 1 can be expressed.

Preferably, anti-human CTLA4 antibody gene and the IL-21 gene of people included in the oncolytic virus genome are α CTLA4-Fc (ALIE)-IL21, nucleotide coding sequence is as shown in SEQID NO:3, the α CTLA4-Fc (ALIE)-of expression IL21 protein amino acid sequence is as shown in SEQID NO:4.

Preferably, the oncolytic virus is to include α CTLA4-Fc (ALIE)-IL21 target gene shown in SEQ ID NO:3 Vaccinia virus recombinant the Temple of Heaven strain, be named as vaccinia virus Tiantan strain rTV- α CTLA4-Fc (ALIE)-IL21, deposit number Are as follows: CCTCC NO:V201934, the deposit date is on Mays 21st, 2019, and preservation address is China typical culture collection center.

Preferably, the anti-human CTLA4 antibody is α CTLA4-Fc (ALIE), it may be assumed that Fc mutant has in anti-human CTLA-4 antibodies There is A330L/I332E to be mutated (ALIE antibody).

Preferably, T2A shearing peptide, P2A can be used between the α CTLA4-Fc (ALIE) and the nucleotide sequence of IL-21 Or IRES is connected, two sections of nucleic acid sequences can be expressed simultaneously in a carrier, and α CTLA4-Fc in two sections of nucleic acid sequences (ALIE) 5 ' ends or 3 ' ends of sequence can be located at；Or two sections of nucleic acid sequence expression of α CTLA4-Fc (ALIE) and IL-21 are in difference Carrier on but be used in combination.

Preferably, the oncolytic virus is by modification to encode and express alpha CTLA4-Fc (ALIE)-IL21 gene, function Energy segment or variant, the gene or its function fragment or variant and SEQ ID NO:3, SEQ ID NO:4 have at least 90% Sequence identity.

Preferably, viral backbone is derived from through modification or modified vaccinia virus Tiantan strain, New York strain, Copenhagen Strain, canary strain, Ankara strain, adenovirus vector, gland relevant viral vector, herpes simplex virus vector, varicella blister Exanthema virus (varicella-zoster virus, VZV) carrier, Respiratory Syncytial Virus(RSV) (Respiratory Syncytial Virus, RSV), Sheng Liji forest virus (Semliki forest virus, SFV), Epstein-Barr virus, cytomegalovirus, people's bleb Viral 6 types, variola virus, vaccinia virus, mollascus contagiosum virus, sheep of virus, reovirus, rotavirus, enteron aisle Virus, Senecan virus, poliovirus, Coxsackie virus, rhinovirus, hepatitis A virus, foot and mouth disease virus, toga Virus, Alphavirus, Semliki Forest virus, Eastern equine encephalitis virus, sindbis alphavirus, rubella virus, coronavirus, Huang Virus, Hepatitis C Virus, japanese encephalitis virus, Saint Louis' encephalitis virus, ink tired paddy fever virus, yellow fever virus, Xi Niluo River virus, zika virus, dengue virus, Ebola virus, Marburg virus, arenavirus, Lassa fever virus, lymphatic Choriomeningitis virus, Pi Qinde virus, Junin virus, machupo virus, Hantaan virus, Rift Valley fever virus, paramyxovirus, Human parainfluenza viruses, mumps virus, monkey virus 5, measles virus, vesicular stomatitis virus, hydrophobin, respiratory syncystial Virus, orthomyxovirus, influenza A virus, influenza B virus, influenza virus C, Hepatitis D virus, simian immunodeficiency defect Virus, 2 type of human immunodeficiency virus type 1 and human immunodeficiency virus, Rous sarcoma virus, thermophilic human T cell leukemia virus 1 Type, MK virus, hepatitis type B virus, Hepatitis E virus, human papilloma virus or polyomavirus.

Preferably, the oncolytic virus skeleton is intracellular mature virus, intracellular packaging virus, cell related packaging disease Malicious or extracellular packaging virus.

Preferably, the preparation method of α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain, including with Lower step:

(1) people α CTLA4-Fc (ALIE)-IL21 is synthesized, gene order is as shown in SEQ ID NO:3；

(2) α CTLA4-Fc (ALIE)-IL21 is subcloned into the area TK of vaccinia virus shuttle plasmid (pSC65), constructs weight Group plasmid pSC65- α CTLA4-Fc (ALIE)-IL21；

(3) by the way of homologous recombination, by pSC65- α CTLA4-Fc (ALIE)-IL21 plasmid transfection to being felt The TK143 of Wild-type vaccinia strain is contaminated^-In cell, makes the two homologous recombination, generate vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21；After screening, obtaining the area TK includes pSC65- α CTLA4-Fc (ALIE)-shown in SEQID NO:3 The recombination oncolytic vaccinia virus of the coded sequence of IL21；Wherein, α CTLA4-Fc (ALIE)-IL21 gene by vaccinia virus morning/ Late promoter p7.5 control.

Preferably, it in the preparation method of α CTLA4-Fc (the ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain, utilizes VERO cell includes: that the growth of VERO cell is close to the specific steps of α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain is instilled when degree is up to close to 100%, replaces low concentration fetal calf serum Maintenance culture medium, every 10 cm culture plate inoculum concentration is about 0.02 MOI oncolytic vaccinia virus, is put into incubator culture, recombinates acne After virus amplification, collection virus liquid after multigelation, carries out density gradient centrifugation with sucrose solution and purifies again.

In addition, oncolytic virus of the present invention can be applied in terms of preparing anti-tumor drug, wherein the tumour is thin selected from B Born of the same parents' lymthoma, t cell lymphoma, melanoma, prostate cancer, clear-cell carcinoma, sarcoma, glioma, High Grade Gliomas, mother are thin Born of the same parents' tumor neuroblastoma, osteosarcoma, plasmacytoma, histocytoma, cancer of pancreas, breast cancer, lung cancer such as Small Cell Lung Cancer With non-small cell lung cancer, gastric cancer, liver cancer, colon and rectum carcinoma, the cancer of the esophagus, colorectal cancer, hemopoietic system cancer, carcinoma of testis, uterine neck Cancer, oophoroma, bladder cancer, squamous cell carcinoma, gland cancer, AIDS associated lymphoma, bladder cancer, the cancer of the brain, nervous system cancer, neck Cancer, head and neck squamous cell carcinoma, hodgkin's lymphomas, non Hodgkin lymphom or blood neoplastic disease.

The invention has the advantages that compared with prior art: the present invention is by the gene therapy of tumour and oncolytic effect phase In conjunction with, be prepared for one kind can high efficient expression human source gene α CTLA4-Fc (ALIE)-IL21 oncolytic virus.By oncolytic virus When dispensing is to tumor microenvironment, it, can also the anti-human CTLA4 of great expression crack tumour cell and play oncolytic effect while Antibody α CTLA4-Fc (ALIE) molecule, to kill the regulatory T-cell in tumor microenvironment, or blocks its depression effect, thus The immune response of tumor microenvironment is raised, the IL-21 of anti-tumor effect, the oncolytic virus while great expression people is promoted, into One step promotes the killing ability of NK cell in tumor microenvironment, plays multiple antitumous effect.It is treated relative to simple gene Method or virus therapy, the oncolytic virus significantly increase the rejection ability to malignant tumour.

In addition, the present invention has completed vaccinia virus Tiantan strain oncolytic virus treatment glioma, melanoma, forefront The preclinical study of gland cancer and pernicious lung cancer etc., realizes the good targeting and antitumous effect to tumour, and have compared with It for complete virus amplification and quality control system, lays the foundation for further industrialization, the present invention has good application Prospect.

Detailed description of the invention

Fig. 1 shows the building of vaccinia virus Tiantan strain shuttle vector (expression mouse IL-21).Fig. 1 a is to be integrated with The vaccinia virus shuttle plasmid pSC65-mIL-21 expression map of mouse IL-21 gene；Fig. 1 b is the vaccinia virus Tiantan strain After infecting VERO cell, the verifying of mouse IL-21 protein expression in culture supernatant.

Fig. 2 shows the effect of the internal melanoma of the vaccinia virus recombinant of mouse IL-21.Fig. 2 a is wild type acne Seedling diseases poison the Temple of Heaven strain (TV) treatment group and mouse IL-21 vaccinia virus recombinant (rTV-mIL-21) treatment group are black to B16 is vaccinated with The control action of the C57BL/6J mice tumors grew of melanoma；Fig. 2 b is Wild-type vaccinia strain the Temple of Heaven strain treatment and mouse The vaccinia virus recombinant of IL-21 treats the influence to the C57BL/6J mouse survival rate for being vaccinated with B16 melanoma.It can from figure Know, the vaccinia virus recombinant of mouse IL-21 can be obviously improved the killing of the mouse tumor to load B16 melanoma in vivo Effect, and the survival rate of load B16 melanoma mouse can greatly be improved.

Fig. 3 shows the internal anti-glioma effect of the vaccinia virus recombinant of mouse IL-21.Fig. 3 a is wild type acne The vaccinia virus recombinant treatment group of seedling diseases poison the Temple of Heaven strain treatment group and mouse IL-21 are to being vaccinated with GL261 nervousness tumor C57BL/6J mouse tumor size control action；Fig. 3 b is the weight of Wild-type vaccinia strain the Temple of Heaven strain treatment group and mouse IL-21 Influence of the group vaccinia virus treatment group to the C57BL/6J mouse survival rate for being vaccinated with GL261 cellular neural matter tumor.It can from figure Know, the vaccinia virus recombinant of mouse IL-21 can be obviously improved the C57BL/6J mouse tumor to GL261 nervousness tumor in vivo Fragmentation effect, and the survival rate of load GL261 nervousness tumor mouse can greatly be improved.

Fig. 4 shows the building and α CTLA4-Fc of human source gene α CTLA4-Fc (ALIE)-IL21 shuttle vector (ALIE) expression of-IL21 albumen.Fig. 4 a is the vaccinia virus shuttle plasmid for being integrated with α CTLA4-Fc (ALIE)-IL21 gene The expression map of pSC65- α CTLA4-Fc (ALIE)-IL21；Fig. 4 b is vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)- The expression of anti-human CTLA4 antibody in supernatant is secreted after IL21 infection VERO cell；Fig. 4 c is vaccinia virus recombinant rTV- α CTLA4- The expression of Fc (ALIE)-IL21 infection VERO cell descendant IL-21.It can be seen that vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE) the anti-human CTLA4 antibody and the equal successful expression of 1 cell factor of human IL-2 of-IL21.

Fig. 5 shows the effect of the internal anti-human lung cancer of vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21.Fig. 5 In be-IL21 couples of control group, NKT cell therapy group and NKT cell union and recombination vaccinia virus rTV- α CTLA4-Fc (ALIE) It is vaccinated with B-NDG (B-NSGTM) mouse tumor size control action of the pernicious lung carcinoma cell of NCI-H292.It can be seen that NKT cell union and recombination vaccinia virus rTV- α CTLA4-Fc (ALIE)-IL21 can be obviously improved in vivo dislikes NCI-H292 The fragmentation effect of property lung cancer.

Fig. 6 shows the internal anti-human prostate cancer of vaccinia virus recombinant rTV- rTV- α CTLA4-Fc (ALIE)-IL21 Effect.It is control group, NKT cell therapy group and NKT cell union and recombination vaccinia virus rTV- α CTLA4-Fc in Fig. 6 (ALIE) the therapeutic effect of-IL21 treatment group to the B-NDG mouse for being vaccinated with LNCaP prostate cancer.It can be seen that NKT is thin Born of the same parents union and recombination vaccinia virus rTV- α CTLA4-Fc (ALIE)-IL21 can be obviously improved in vivo to LNCaP prostate cancer Fragmentation effect.

Preservation information: vaccinia virus Tiantan strain rTV- α CTLA4-Fc (ALIE)-IL21, Latin name areOrthopoxvirus genus, China typical culture collection center is deposited on May 21st, 2019, address is located at China Wuhan, Wuhan University, postcode 430072, deposit number are as follows: CCTCC NO:V201934.

Specific embodiment

Embodiment one: the building and expression verifying of the vaccinia virus recombinant rTV-mIL-21 of mouse IL-21

The 1.1 pSC65 vector constructions with mouse IL-21 target gene

The DNA sequence dna of artificial synthesized mouse source IL-21, the sequence of synthesis is as shown in SEQ ID NO:1, and amino acid sequence is such as Shown in SEQ ID NO:2, PCR amplification is carried out using following primer using the DNA sequence dna of synthesis as template.

The primer of amplification are as follows:

Mouse source IL-21-F:GTACCAGGCCTAGTACTATGGAGAGGACCCTTGTCTG

Mouse source IL-21-R:AATAAGCTCGAAGTCGAC CTAGGAGAGATGCTGATG

PCR response procedures: 94 DEG C initial denaturation 5 minutes；98 DEG C are denaturalized 10 seconds, 58 DEG C: annealing 30 seconds, 72 DEG C extend 1 point Clock reacts 30 circulations；72 DEG C of rings sufficiently extend 10 minutes again, terminate at 25 DEG C.

The recycling of PCR product and clone construct: after amplification, target gene are separated in 2% Ago-Gel, together When by pSC65 carrier Sal I digestion (Thermo Scientific company, article No. ER0642) linearized vector, and cut Glue recycling, using Sanprep pillar DNA plastic recovery kit (Promega company, article No. A9282) recycling PCR segment section with Carrier endonuclease bamhi.Gene recovery product connect (Nuo Weizan company, article No. with the method for linearization for enzyme restriction carrier homologous recombination C112-02).Connection product is converted to E. coli TOP10, the overnight growth on the culture plate of the mycin of benzyl containing ammonia. 2nd day, random picking single colonie was sequenced, and mutational site correction, after verifying full sequence is correct, successful clone goes out mouse The pSC65 shuttle plasmid (pSC65-mIL-21) of source IL-21 gene, plasmid construction map are as shown in Figure 1a.

The recombination of the vaccinia virus recombinant of 1.2 mouse IL-21

1. cell prepares: by 143TK^-Cell is layered in 6 orifice plates, every hole about 1 × 10⁶It is a.Culture 24 hours or so, works as cell It is adherent and when being paved with entire bottom surface, carry out next step operation.

2. vaccinia virus is incubated for: with 0.0125/3 PFU(PFU: plaque forming unit, virus liquid titre)/cell it is wild Type vaccinia virus Tiantan strain infection cell, 37 DEG C of incubators take out after being incubated for 1 hour, sop up supernatant, and rinse one with 1 mL PBS All over adding 1 mL complete medium.

3. plasmid transfection: the shuttle plasmid pSC65-mIL-21 with mouse IL-21 is transfected 143TK^-Cell.37 DEG C incubate Case culture 48 hours or so, the specific time was depending on cytopathy situation.

4. 2 × the DMEM for preparing virus paving spot maintains culture medium (containing 2%PS and 4%FBS), the low melting point of 2% preheating is added Agarose adds the final concentration of 200 μ g/mL of X-gal(again).

5. sopping up the supernatant in 6 orifice plates, the mixture of 4 mL paving spot is added in 6 orifice plates, every 300 μ L of hole.Then small The heart is put into 4 DEG C of refrigerators, promotes solidification, is transferred in 37 DEG C of incubators after low melting-point agarose solidification again and is inverted culture until occurring clear Clear locus coeruleus.

6. the viral locus coeruleus of picking recombination, is added the complete medium of 500 μ L.- 80 DEG C of multigelations more than three times, The release for keeping virus more as far as possible.

7. by 143TK^-Cell is layered in 6 orifice plates, every hole about 1 × 10⁶It is a.Culture 24 hours or so, until cell is adherent And it is paved with entire bottom surface.

8. blowing and beating the locus coeruleus in EP pipe repeatedly, it is made to scatter completely.

Maintain culture medium that then the virus liquid containing locus coeruleus is added 9. complete medium is changed into, 37 DEG C of incubators are incubated for 3- 4 hours.

10. screening pressure is added: BrdU working concentration is 50 μ g/mL, is put into 37 DEG C of incubators and is incubated for 48 hours or so, Paving spot is carried out according to viral plaque formational situation.The purification process at least needs to carry out 5 times.

11. then carrying out the sample amplification of vaccinia virus recombinant, 143TK is spread^-Cell is in six orifice plates, every hole 1 × 10⁶It is a thin Born of the same parents, cell is about the 100% of orifice plate floor space when use.

12. changing the culture medium in hole into 2 mL before kind of poison maintains culture medium.The virus containing locus coeruleus that purifying is obtained Liquid is blown and beaten to locus coeruleus repeatedly to scatter.100 μ L or so virus liquid is added in every hole.37 DEG C of incubators are incubated for 48 hours or so, according to disease Malicious spot formational situation receives sample.

13. receiving sample: 1 mL is carefully sucked out in the culture medium supernatant in hole.Cell is sufficiently blown with remaining 1 mL culture medium Under, it closes in EP pipe, can be used for the extraction of subsequent gene group and expanded as seed culture of viruses.

The expression of the vaccinia virus recombinant (rTV-mIL-21) of 1.3 mouse IL-21 is verified

1. taking 10 cm wares, about 5 × 10 are inoculated in each ware⁶A VERO cell guarantees to make when second day inoculation vaccinia virus Cell density is advisable up to 100%；

2. before virus inoculation, complete medium need to be changed into 8 mL and maintain culture medium (DMEM culture medium+2%FBS+1%PS), made Cell is no longer grown, and utilizes the amplification of virus；By virus inoculation into the cell for maintaining culture medium, inoculum concentration is about 0.02 MOI(MOI=virus PFU/ cell number).

3. cultivating 48 hours or so in the incubator of 37 DEG C of 5% CO2, the metainfective VERO cell an of ware is taken, is received Take cell, after with PBS wash cell twice, collect metainfective VERO cell, mouse analyzed by protein immunoblotting method The expression of source IL-21.Primary antibody used is respectively Anti-IL-21 mAb (Invitrogen company, article No. 71003), and two Anti- is respectively HRP label goat antirabbit (company, Zhong Shan Golden Bridge, article No. zb-2301).The results show that recombination has mouse source IL- After 21 vaccinia virus infection VERO cell, mouse source IL-21 albumen can be detected using protein immunoblotting method Height expression, infection the Temple of Heaven vaccinia virus wild strain also have a small amount of expression (Fig. 1 b).

Embodiment two: the melanoma effect of the vaccinia virus recombinant of mouse IL-21

The amplification and purifying of the vaccinia virus recombinant of 2.1 mouse IL-21

1.VERO plating cells: 10 cm wares, each ware about 5 × 10⁶A cell guarantees to make when second day inoculation vaccinia virus thin Born of the same parents' density is advisable up to 100%；

2. before virus inoculation, complete medium need to be changed into 8 mL and maintain culture medium (DMEM culture medium+2%FBS+1%PS), it will For virus inoculation into the cell for maintaining culture medium, inoculum concentration is about 0.02 MOI(MOI=virus PFU/ cell number).Continue 37 It is cultivated 48 hours or so in the incubator of DEG C 5% CO2, sample is received according to viral plaque formational situation；

3. receiving vaccinia virus: discarding 8 mL culture medium in ware, take 2 mL and culture medium is maintained to blow down remaining cell, close at 15 In mL centrifuge tube；

4. after freezing 24 hours, by the virus liquid received multigelation 2 times again, with 36% sucrose solution carry out density gradient from The heart, 16,000 4 DEG C of g are centrifuged 90 min, carefully outwell supernatant, with the viral pellet in PBS buffer solution dissolution centrifuge tube, packing - 80 DEG C are stored in, virus titer to be determined.

The titer determination of the vaccinia virus recombinant of 2.2 mouse IL-21

1.143TK^-The preparation of cell: by 143TK^-Cell is layered in 24 orifice plates, and every hole is about 2 × 10⁵A cell, when use, are thin Born of the same parents' density will reach the 100% of 24 orifice plate floor spaces；

2. dilution virus dilutes vaccinia virus with maintenance culture medium(rTV-mIL-21)Virus liquid does 10 times since 1:100 Than dilution, final volume is 1100 μ L；

3. discarding the complete medium in 24 orifice plates, takes in the 500 μ L adding hole of virus liquid diluted, do two multiple holes.37℃ 5% CO₂Incubator in be incubated for 48 hours or so, the paving spot time is determined according to the thermophilic spot formational situation of virus；

4. paving spot method: preparing paving spot culture medium and 8 mL boiling water baths that 8 mL contain 2 × DMEM culture medium+4%FBS+2%PS The low melting-point agarose being also placed in 37 DEG C of water-baths after thawing, the two is mixed, then X-gal is added in mixture, dense eventually Degree is 200 μ g/mL, for use；

5. sopping up the supernatant in 24 orifice plates.Paving spot mixture in step 4 is added immediately in 24 orifice plates, every 500 μ L of hole.Then 4 DEG C of refrigerators are carefully placed into, solidification is promoted, is transferred to again after low melting-point agarose solidification in 37 DEG C of incubators and is inverted culture until occurring Clearly locus coeruleus；

6. viral plaque counts: whether the number for looking first at the thermophilic spot of virus is in that the trend of ten multiple proportions is successively decreased, then statistics kind poison Only has the quantity of units locus coeruleus in two multiple holes, the sum of locus coeruleus numerical value, dilutes multiplied by corresponding to the hole in two obtained holes The reciprocal value of degree is titre viral in 1 mL.

The internal melanoma effect of the vaccinia virus recombinant of 2.3 mouse IL-21

1. (mouse passes through subcutaneous implantation 7 × 10 to pair C57BL/6J mouse⁴Melanoma B16 cell (125 μ L), daily record The long and short diameter of the tumour of mouse, is calculated using the following equation the volume of tumour.

2. gross tumor volume calculation formula: gross tumor volume (mm³)=(major diameter × wide diameter²)/2.

3. 10 days after mouse inoculation Melanoma B16 cell, the mouse of tumor formation is randomly divided into two groups (every group 5 small Mouse), respectively the strain of the Wild-type vaccinia strain the Temple of Heaven (TV) treatment group, mouse IL-21 vaccinia virus recombinant (rTV-mIL-21) Treatment group.Administration mode is to feed back in tumor, and single-dose is primary.

A:TV group: 1 × 10⁷Every mouse of PFU；

B:rTV-mIL-21 group: 1 × 10⁷Every mouse of PFU.

4. tumor growth curve monitors: after feeding back cell, monitoring tumor size daily using vernier caliper, monitor duration 30 days.Using the long diameter and wide diameter diameter of vernier caliper measurement knurl, gross tumor volume is calculated.

5. mouse survival curve monitors: according to the regulation of Animal Experimental Ethical, when certain mouse tumor diameter is in any side To 2 cm are had more than, the euthanasia of mouse is just carried out, which is denoted as death, monitors duration 88 days.

As a result as shown, compared to Wild-type vaccinia strain the Temple of Heaven strain treatment group, the vaccinia virus recombinant of mouse IL-21 Treatment group, treatment group can significantly control the growth of C57BL/6J murine melanoma, there is the tumour body of 3 mouse in 5 mouse Product control is in 100 mm³Hereinafter, even all removing (Fig. 2 a).And the vaccinia virus recombinant treatment group treatment of mouse IL-21 Group can significantly improve the survival rate (Fig. 2 b) of load melanoma mouse.

Embodiment three: anti-glioma effect in the vaccinia virus recombinant body of mouse IL-21

1. pair C57BL/6 mouse passes through subcutaneous implantation 1 × 10⁶Glioma GL261 cell (125 25), daily record The long and short diameter of the tumour of mouse, is calculated using the following equation the volume of tumour.

3. 7 days after mouse inoculation glioma GL261 cell, the mouse of tumor formation is randomly divided into three groups (every group 5 Mouse), respectively control group, the strain of the Wild-type vaccinia strain the Temple of Heaven (TV) treatment group and mouse IL-21 vaccinia virus recombinant (rTV-mIL-21) treatment group.Oncolytic virus administration mode is intratumor injection feedback, and is derived from the oncolytic disease purified in example two Poison, single-dose are primary.

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

B:TV group: 1 × 10⁷Every mouse of PFU；

C:rTV-mIL-21 group: 1 × 10⁷Every mouse of PFU.

As a result as shown, compared to Wild-type vaccinia strain the Temple of Heaven strain treatment group, the vaccinia virus recombinant of mouse IL-21 Treatment group, treatment group can significantly control the growth of C57BL/6J mouse glioma, there is the swollen of 4 mouse in 5 mouse Tumor fixing fabric structure is in 100 mm³Hereinafter, even all removing (Fig. 3 a).And the vaccinia virus recombinant treatment group of mouse IL-21 Treatment group can significantly improve the survival rate (Fig. 3 b) of load glioma mouse.

Example IV: the building and expression verifying of vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21

4.1 construct the shuttle plasmid of α CTLA4-Fc (ALIE)-IL21 using carrier pSC65

The DNA sequence dna of artificial synthesized human source gene α CTLA4-Fc (ALIE)-IL21 is connected with T2A sequence between two genes, The sequence of synthesis is as shown in SEQ ID NO:3, and amino acid sequence is as shown in SEQ ID NO:4, using the DNA sequence dna of synthesis as mould Version carries out PCR amplification using following primer.

The primer of amplification are as follows:

α CTLA4-Fc (ALIE)-IL21-F:GTACCAGGCCTAGTACTATGGGCTGGTCTTGCATTAT

α CTLA4-Fc (ALIE)-IL21-R:AATAAGCTCGAAGTCGACTCAGGAATCTTCACTTCCGTGTGT

PCR response procedures: 94 DEG C initial denaturation 5 minutes；98 DEG C are denaturalized 10 seconds, 58 DEG C: annealing 30 seconds, 72 DEG C extend 2 points Clock reacts 30 circulations；72 DEG C of rings sufficiently extend 10 minutes again, terminate at 25 DEG C.

The recycling of PCR product and clone construct: after amplification, target gene are separated in 2% Ago-Gel, together When by pSC65 carrier Sal I digestion (Thermo Scientific company, article No. ER0642) gel extraction, use Sanprep pillar DNA plastic recovery kit (Promega company, article No. A9282) recycles PCR segment section and carrier digestion piece Section, gene recovery product connect (Nuo Weizan company, article No. c112-02) with the method for linearization for enzyme restriction carrier homologous recombination, Connection product is converted to E. coli TOP10, the overnight growth on the culture plate of the mycin of benzyl containing ammonia.2nd day, with Machine picking single colonie is sequenced, and mutational site correction, after verifying full sequence is correct, successful clone goes out α CTLA4-Fc (ALIE) the pSC65 carrier (pSC65- α CTLA4-Fc (ALIE)-IL21) of-IL21 gene, plasmid construction map such as Fig. 4 a.

4.2 building vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21

2. vaccinia virus is incubated for: with the Wild-type vaccinia strain the Temple of Heaven strain infection cell of 0.0125/3 (PFU/ cell), 37 DEG C incubator takes out after being incubated for 1 hour, sops up supernatant, and adds 1 mL complete medium one time with 1 mL PBS flushing.

3. plasmid transfection: above-mentioned shuttle plasmid pSC65- α CTLA4-Fc (ALIE)-IL21 is transfected 143TK^-Cell.37 DEG C incubator culture 48 hours or so, the specific time was depending on cytopathy situation.

6. picking locus coeruleus (containing there is purpose vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21) is added 500 μ L's Complete medium.- 80 DEG C of multigelations more than three times, the release for keeping virus more as far as possible.

The expression of 4.3 vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21 is verified

1. taking 10 cm culture dishes, inoculation 5 × 10⁶A VERO cell/ware guarantees to keep cell close when second day inoculation vaccinia virus Degree is advisable up to 100%.Before virus inoculation, complete medium need to be changed into 8 mL and maintain culture medium (DMEM culture medium+2%FBS+1% PS)；Then virus is added, inoculum concentration is about 0.02 MOI.It is cultivated 24 hours in the incubator of 37 DEG C of 5% CO2.

2. taking above-mentioned metainfective VERO cell, a part, which is fixed, wears film, and room temperature is protected from light 20min.Reaction knot The cleaning solution of 800 μ l is added after beam, after mixing, 12000 g, 30 s centrifugation, wash 1-2 times, abandon supernatant, after washed carefully with PBS Born of the same parents twice, then with the antibody (BD Pharminge company, model 560463) of anti-IL-21-PE are dyed, finally with stream Formula cell art (BD Pharminge company, model forttesa) analysis.The results show that vaccinia virus recombinant rTV- α CTLA4- Fc (ALIE)-IL-21 can express target gene human IL-2 1(Fig. 4 b in VERO cell).

3. simultaneously, remaining VERO cell continuation is cultivated 48 hours in the incubator of 37 DEG C of 5% CO2, according to viral plaque Formational situation collects cell conditioned medium, is purified with Protein G agarose column (GE company, article No. 28-9031-34), pure Antibody after change is dissolved with PBS buffer solution.As illustrated in fig. 4 c, anti-human CTLA4 antibody after purification can be with the Treg of expression CTLA4 Cell combination.

Embodiment five: the anti-human lung cancer effect of vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21

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

2. before virus inoculation, complete medium need to be changed into 8 mL and maintain culture medium (DMEM culture medium+2%FBS+1%PS), made Cell is no longer grown, and utilizes the amplification of virus；By virus inoculation into the cell for maintaining culture medium, inoculum concentration is about 0.02 MOI.Continuation is cultivated 48 hours or so in the incubator of 37 DEG C of 5% CO2, receives sample according to viral plaque formational situation；

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

2. dilution virus does 10 doubling dilutions, final volume since 1:100 with maintaining culture medium to dilute vaccinia virus virus liquid For 1100 μ L；

The effect of the anti-human lung cancer of 5.3 vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21

1. pair B-NDG mouse passes through subcutaneous implantation 5 × 10⁶Human lung carcinoma cell (NCI-H292,125 μ L), record mouse daily The long and short diameter of tumour, be calculated using the following equation the volume of tumour.

According to the regulation of Animal Experimental Ethical, when certain mouse tumor diameter has more than 2 cm in any direction, just carry out The euthanasia of mouse, the experiment mice are denoted as dead (it is expected that tumor formation in 10 days or so).

3. 30 days after mouse inoculation tumour NCI-H292 cell, the mouse of tumor formation is randomly divided into four groups (every group 5 small Mouse), a respectively untreated control group, two treatment groups include NKT cell therapy group (NKT group), NKT cell joint weight Group vaccinia virus rTV- α CTLA4-Fc (ALIE)-IL21 treatment group (NKT+rTV group).Administration mode is intratumor injection feedback, single It is secondary to be administered once.

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

B:NKT group: 5 × 10⁶A NKT cell；

C:NKT+ rTV group: 5 × 10⁶A NKT cell joint 1 × 10⁶The vaccinia virus recombinant rTV- α CTLA4-Fc of PFU (ALIE)-IL21；

4. tumor growth curve monitors: after feeding back cell, monitoring tumor size daily using vernier caliper, monitor duration 15 days. Using the long diameter and wide diameter diameter of vernier caliper measurement knurl, gross tumor volume is calculated.

As a result such as Fig. 5 is shown, compared to untreated control group, NKT cell therapy group can a degree of control B-NDG The growth of mouse NCI-H292 tumor, but observe the 15th day after inoculated tumour cell, the tumour body of NKT cell therapy group mouse Product is more than 600 mm³；And NKT cell union and recombination vaccinia virus rTV- α CTLA4-Fc (ALIE)-IL21 treatment group, equal energy The growth of enough significant control B-NDG mouse NCI-H292 lung cancer, the gross tumor volume of mouse can be controlled in 100 mm³Hereinafter, Even all remove.

Embodiment six: the anti-human prostate cancer effect of vaccinia virus recombinant rTV- α CTLA4-Fc (ALIE)-IL21

1. pair B-NDG mouse passes through subcutaneous implantation 5 × 10⁶Prostate gland cancer cell (LNCaP, 125 μ L), record mouse daily The long and short diameter of tumour, be calculated using the following equation the volume of tumour.

3. 20 days after mouse inoculation tumour LNCaP cell, the mouse of tumor formation is randomly divided into three groups (every group of 5 mouse), A respectively untreated control group, two treatment groups include NKT cell therapy group (NKT group), NKT cell union and recombination acne Seedling diseases poison rTV- α CTLA4-Fc (ALIE)-IL21 treatment group (NKT+rTV group).Oncolytic virus administration mode is returned for intratumor injection Defeated, single-dose is primary.

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

B:NKT group: 5 × 10⁶A NKT cell；

4. tumor growth curve monitors: after feeding back cell, monitoring tumor size daily using vernier caliper, monitor duration 30 days. Using the long diameter and wide diameter diameter of vernier caliper measurement knurl, gross tumor volume is calculated.

As a result as Fig. 6 shows that, compared to control group, NKT treatment group can be in a degree of control B-NDG mouse LNCaP The growth of tumor, the 15th day after inoculated tumour cell, 5 mouse tumor sizes in NKT group are in 1000 mm³Below.But it observes The 30th day after to inoculated tumour cell, the gross tumor volume of NKT cell therapy group mouse was more than 1000 mm³；And NKT cell Union and recombination vaccinia virus rTV- α CTLA4-Fc (ALIE)-IL21 treatment group can significantly control B-NDG mouse LNCaP tumor Growth, can by the gross tumor volume of mouse control in 100 mm³Hereinafter, even 5 mouse tumors are all removed.

In conclusion the present invention combines the gene therapy of tumour with oncolytic effect, building includes anti-human CTLA4 antibody With the gene coded sequence of human IL-2 1, and the novel oncolytic virus of anti-human CTLA4 antibody and human IL-2 1, viral bone can be expressed Frame is derived from through modification or modified vaccinia virus Tiantan strain, New York strain, Copenhagen strain, canary strain, Ankara strain, gland Viral vectors, gland relevant viral vector, herpes simplex virus vector, varicella virus (varicella-zoster Virus, VZV) carrier, Respiratory Syncytial Virus(RSV) (Respiratory Syncytial Virus, RSV), Sheng Liji Forest Diseases Malicious (Semliki forest virus, SFV), Epstein-Barr virus, cytomegalovirus, human herpes virus type 6, variola virus, bovine vaccine disease Poison, mollascus contagiosum virus, sheep of virus, reovirus, rotavirus, enterovirus, Senecan virus, gray nucleus Scorching virus, Coxsackie virus, rhinovirus, hepatitis A virus, foot and mouth disease virus, togavirus, Alphavirus, Semliki Forest Virus, Eastern equine encephalitis virus, sindbis alphavirus, rubella virus, coronavirus, flavivirus, Hepatitis C Virus, Japanese brain Scorching virus, Saint Louis' encephalitis virus, ink tire out paddy fever virus, yellow fever virus, West Nile Virus, zika virus, dengue virus, Ebola virus, Marburg virus, arenavirus, Lassa fever virus, lymphocytic choriomeningitis virus, Pi Qinde disease Poison, Junin virus, machupo virus, Hantaan virus, Rift Valley fever virus, paramyxovirus, human parainfluenza viruses, mumps virus, Monkey virus 5, measles virus, vesicular stomatitis virus, hydrophobin, Respiratory Syncytial Virus(RSV), orthomyxovirus, Flu-A disease Poison, influenza B virus, influenza virus C, Hepatitis D virus, monkey immunodeficiency virus, human immunodeficiency virus type 1 and 2 type of human immunodeficiency virus, Rous sarcoma virus, 1 type of thermophilic human T cell leukemia virus, MK virus, hepatitis B The multiplexed virals such as poison, Hepatitis E virus, human papilloma virus or polyomavirus.It can be used in controlling human lung cancer and prostate Cancer, B cell lymphoma, t cell lymphoma, melanoma, prostate cancer, clear-cell carcinoma, sarcoma, glioma, high-level colloid Tumor, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histocytoma, cancer of pancreas, breast cancer, lung cancer are such as small thin Born of the same parents' lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon and rectum carcinoma, the cancer of the esophagus, colorectal cancer, hemopoietic system cancer, carcinoma of testis, Cervical carcinoma, oophoroma, bladder cancer, squamous cell carcinoma, gland cancer, AIDS associated lymphoma, bladder cancer, the cancer of the brain, nervous system cancer, head A variety of entities such as neck cancer, head and neck squamous cell carcinoma, hodgkin's lymphomas, non Hodgkin lymphom or blood neoplastic disease Tumour has very high application value for the treatment of tumour and preparation is simple, convenient for a large amount of preparations and promotes the use of.

The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent thereof.

Sequence table

<110>Shanghai Public Health Clinical Center

<120>a kind of novel oncolytic virus and its preparation method and application

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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

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<212> PRT

<213>artificial sequence (Artificial Sequence)

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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

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<213>artificial sequence (Artificial Sequence)

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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 kind of novel oncolytic virus, which is characterized in that include anti-human CTLA4 antibody and people in the oncolytic virus genome The gene coded sequence of IL-21, and anti-human CTLA4 antibody and human IL-2 1 can be expressed.

2. a kind of novel oncolytic virus according to claim 1, which is characterized in that included in the oncolytic virus genome Anti-human CTLA4 antibody gene and people IL-21 gene be α CTLA4-Fc (ALIE)-IL21, nucleotide coding sequence is such as Shown in SEQID NO:3, α CTLA4-Fc (the ALIE)-IL21 protein amino acid sequence of expression is as shown in SEQID NO:4.

3. a kind of novel oncolytic virus according to claim 1, which is characterized in that the oncolytic virus is to include SEQ ID The vaccinia virus recombinant the Temple of Heaven strain of α CTLA4-Fc shown in NO:3 (ALIE)-IL21 target gene, is named as vaccinia virus Tiantan strain RTV- α CTLA4-Fc (ALIE)-IL21, deposit number are as follows: CCTCC NO:V201934, the deposit date is Mays 21 in 2019 Day, preservation address is China typical culture collection center.

4. a kind of novel oncolytic virus according to claim 1, which is characterized in that the anti-human CTLA4 antibody is α CTLA4- Fc (ALIE), it may be assumed that Fc mutant has A330L/I332E mutation (ALIE antibody) in anti-human CTLA-4 antibodies.

5. a kind of novel oncolytic virus according to claim 3, which is characterized in that the α CTLA4-Fc (ALIE) and IL-21 Nucleotide sequence between T2A shearing peptide can be used, P2A or IRES are connected, two sections of nucleic acid sequences can be same in a carrier When express, and in two sections of nucleic acid sequences α CTLA4-Fc (ALIE) can be located at sequence 5 ' end or 3 ' end；Or α CTLA4-Fc (ALIE) it on different carriers but is used in combination from two sections of nucleic acid sequence expression of IL-21.

6. a kind of novel oncolytic virus according to claim 2, which is characterized in that the oncolytic virus is by modification to encode With express alpha CTLA4-Fc (ALIE)-IL21 gene, function fragment or variant, the gene or its function fragment or variant with SEQ ID NO:3, SEQ ID NO:4 have at least 90% sequence identity.

7. a kind of novel oncolytic virus according to claim 1, which is characterized in that its viral backbone derives from and modified or passed through The vaccinia virus Tiantan strain of transformation, New York strain, Copenhagen strain, canary strain, Ankara strain, adenovirus vector, gland related diseases Poisonous carrier, varicella virus (varicella-zoster virus, VZV) carrier, is exhaled at herpes simplex virus vector Inhale road syncytial virus (Respiratory Syncytial Virus, RSV), Sheng Liji forest virus (Semliki forest Virus, SFV), Epstein-Barr virus, cytomegalovirus, human herpes virus type 6, variola virus, vaccinia virus, mollascus contagiosum virus, Sheep of virus, reovirus, rotavirus, enterovirus, Senecan virus, poliovirus, Coxsackie virus, Rhinovirus, hepatitis A virus, foot and mouth disease virus, togavirus, Alphavirus, Semliki Forest virus, Eastern Equine Encephalitis disease Poison, sindbis alphavirus, rubella virus, coronavirus, flavivirus, Hepatitis C Virus, japanese encephalitis virus, St. Louis brain Scorching virus, ink tired paddy fever virus, yellow fever virus, West Nile Virus, zika virus, dengue virus, Ebola virus, Marburg Virus, arenavirus, Lassa fever virus, lymphocytic choriomeningitis virus, Pi Qinde virus, Junin virus, Ma Qiu Wave virus, Hantaan virus, Rift Valley fever virus, paramyxovirus, human parainfluenza viruses, mumps virus, monkey virus 5, measles virus, Vesicular stomatitis virus, hydrophobin, Respiratory Syncytial Virus(RSV), orthomyxovirus, influenza A virus, influenza B virus, Influenza virus C, Hepatitis D virus, monkey immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus 2 Type, Rous sarcoma virus, 1 type of thermophilic human T cell leukemia virus, MK virus, hepatitis type B virus, Hepatitis E virus, Human papilloma virus or polyomavirus.

8. novel oncolytic virus according to claim 1, which is characterized in that the oncolytic virus skeleton is intracellular mature Viral, intracellular packaging virus, cell related packaging virus or extracellular packaging virus.

9. the preparation method of α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain as claimed in claim 3, including with Lower step:

10. the amplification method of α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain according to claim 9, It is characterized in that, includes: using specific steps of the VERO cell to α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain α CTLA4-Fc (ALIE)-IL21 vaccinia virus recombinant the Temple of Heaven strain, replacement are instilled when VERO cell density is reached close to 100% The maintenance culture medium of low concentration fetal calf serum, every 10 cm culture plate inoculum concentration is about 0.02 MOI oncolytic vaccinia virus, is put into Incubator culture after recombinant poxvirus expands, collects virus liquid again after multigelation, with sucrose solution carry out density gradient from Heart purifying.

11. application of the novel oncolytic virus in terms of preparing anti-tumor drug described in claim 1.

12. application of the novel oncolytic virus according to claim 11 in terms of preparing anti-tumor drug, wherein described swollen Tumor is selected from B cell lymphoma, t cell lymphoma, melanoma, prostate cancer, clear-cell carcinoma, sarcoma, glioma, high-level glue Matter tumor, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histocytoma, cancer of pancreas, breast cancer, lung cancer are such as small Cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon and rectum carcinoma, the cancer of the esophagus, colorectal cancer, hemopoietic system cancer, testis Cancer, cervical carcinoma, oophoroma, bladder cancer, squamous cell carcinoma, gland cancer, AIDS associated lymphoma, bladder cancer, the cancer of the brain, nervous system Cancer, head and neck cancer, head and neck squamous cell carcinoma, hodgkin's lymphomas, non Hodgkin lymphom or blood neoplastic disease.