CN110205307A - 去除vgf基因的重组天坛株溶瘤痘苗病毒及制备和应用 - Google Patents
去除vgf基因的重组天坛株溶瘤痘苗病毒及制备和应用 Download PDFInfo
- Publication number
- CN110205307A CN110205307A CN201910012884.5A CN201910012884A CN110205307A CN 110205307 A CN110205307 A CN 110205307A CN 201910012884 A CN201910012884 A CN 201910012884A CN 110205307 A CN110205307 A CN 110205307A
- Authority
- CN
- China
- Prior art keywords
- temple
- vaccinia virus
- virus
- recombination
- oncolytic vaccinia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101150004676 VGF gene Proteins 0.000 title claims abstract description 63
- 241000700618 Vaccinia virus Species 0.000 title claims abstract description 62
- 230000000174 oncolytic effect Effects 0.000 title claims abstract description 57
- 230000006798 recombination Effects 0.000 title claims abstract description 43
- 238000005215 recombination Methods 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims abstract description 16
- 229960005486 vaccine Drugs 0.000 claims abstract description 5
- 241000700605 Viruses Species 0.000 claims description 53
- 230000003612 virological effect Effects 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 15
- 239000000725 suspension Substances 0.000 claims description 7
- 241000700649 Vaccinia virus Tian Tan Species 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- 230000006801 homologous recombination Effects 0.000 claims description 4
- 238000002744 homologous recombination Methods 0.000 claims description 4
- 239000003550 marker Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 206010067268 Post procedural infection Diseases 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 3
- 239000013553 cell monolayer Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000000799 fluorescence microscopy Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000001890 transfection Methods 0.000 claims description 3
- 238000002525 ultrasonication Methods 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 230000009385 viral infection Effects 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 238000001815 biotherapy Methods 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 201000007270 liver cancer Diseases 0.000 abstract description 2
- 208000014018 liver neoplasm Diseases 0.000 abstract description 2
- 201000005202 lung cancer Diseases 0.000 abstract description 2
- 208000020816 lung neoplasm Diseases 0.000 abstract description 2
- 201000001441 melanoma Diseases 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 210000002845 virion Anatomy 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 23
- 210000004881 tumor cell Anatomy 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000002147 killing effect Effects 0.000 description 5
- 231100000225 lethality Toxicity 0.000 description 5
- 239000000284 extract Substances 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 208000002874 Acne Vulgaris Diseases 0.000 description 3
- 206010000496 acne Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001018 virulence Effects 0.000 description 3
- 238000011729 BALB/c nude mouse Methods 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 210000005265 lung cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- HDOVUKNUBWVHOX-QMMMGPOBSA-N Valacyclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCOC(=O)[C@@H](N)C(C)C)C=N2 HDOVUKNUBWVHOX-QMMMGPOBSA-N 0.000 description 1
- 241000700647 Variola virus Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000309459 oncolytic virus Species 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 230000029305 taxis Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种去除VGF基因的重组天坛株溶瘤痘苗病毒,去除天坛株溶瘤痘苗病毒VGF基因,表达EGFP基因,其中VGF基因的碱基序列如序列表1所示。本发明还公开了该去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法及应用。本发明去除VGF基因的重组天坛株溶瘤痘苗病毒VTT‑VGFE,降低了天坛株溶瘤痘苗病毒的毒性,提高了天坛株溶瘤痘苗病毒的肿瘤选择性,且具有实时监测病毒颗粒在体内分布的功能,为痘苗病毒作为载体研发活疫苗及肿瘤生物治疗提供了良好工具,能够适用于治疗肺癌、肝癌、黑色素瘤等多种肿瘤。
Description
技术领域
本发明属于生物基因工程技术领域,涉及一种去除VGF基因的重组天坛株溶瘤痘苗病毒,本发明还涉及一种去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法和应用。
背景技术
肿瘤生物治疗技术是近年来兴起的一种全新的肿瘤治疗手段,具有副作用小、抗肿瘤活性高、适应性广等特点。
痘苗病毒(vaccinia virus,VACV)作为溶瘤病毒对肿瘤具有天然的趋向性,可选择性的感染肿瘤细胞,并在其内复制,不会被免疫***很快清除。痘苗病毒天坛株(TianTan strain,VTT)是我国特有的一株痘苗病毒,作为人类预防天花的疫苗,曾经在全世界范围内普遍使用,因此痘苗病毒为癌症治疗提供了新的方向。为了进一步提高痘苗病毒对肿瘤细胞的选择性,需要对病毒进行一定的改造。病毒生长因子(virus growth factor,VGF)是一种与表皮生长因子同源的分泌型蛋白,可与细胞表面的表皮生长因子受体结合从而促进细胞增殖,这对病毒在正常组织中复制具有重要意义,但在肿瘤细胞中却可有可无,去除VTT的VGF基因可增强肿瘤选择性,提高病毒安全性。但是目前尚缺乏去除VGF基因的减毒天坛株病毒。
发明内容
本发明的目的是提供一种去除VGF基因的重组天坛株溶瘤痘苗病毒VTT-VGFE,能够降低天坛株溶瘤痘苗病毒的毒性,提高天坛株溶瘤痘苗病毒的肿瘤选择性。
本发明还提供了去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法。
及应用
本发明所采用的技术方案是,去除VGF基因的重组天坛株溶瘤痘苗病毒,去除天坛株溶瘤痘苗病毒VGF基因,其中VGF基因的碱基序列如序列表1所示。
本发明采用的第一种技术方案的特点在于:
重组天坛株溶瘤痘苗病毒中***了绿色荧光蛋白基因EGFP,绿色荧光蛋白基因EGFP由启动子子P7.5控制表达,绿色荧光蛋白基因EGFP位于重组天坛株溶瘤痘苗病毒的VGF区,绿色荧光蛋白基因EGFP的碱基序列如序列表2所示。
重组天坛株溶瘤痘苗病毒的出发病毒为天坛株。
本发明采用的另一种技术方案是:
去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法,具体按照下述步骤进行:
步骤1,将针对VTT病毒VGF基因的打靶质粒与VTT病毒在细胞内重组后,收集病毒;
步骤2,筛选纯化重组病毒,得到去除VGF基因的重组天坛株溶瘤痘苗病毒。
本发明另一种技术方案的特点还在于:
步骤1中针对VTT病毒VGF基因的打靶质粒为pMV-VTTVGFE,打靶质粒pMV-VTTVGFE含有出发载体pMV-Pro、痘苗病毒天坛株VGF基因的上游同源重组臂VTT-VGFL和下游同源重组臂VTT-VGFR,在VTT-VGFL与VTT-VGFR之间有荧光标记基因EGFP和1个多克隆位点,其中荧光标记基因EGFP由启动子P7.5控制表达,多克隆位点处包含启动子P11。
步骤1具体按照下述步骤进行:
步骤1.1,在T25细胞培养瓶中培养CV-1单层细胞,至70~80%覆盖;
步骤1.2,用VTT病毒感染CV-1细胞,在37℃的温度下培养2~3小时;
步骤1.3,将针对VTT病毒VGF基因的打靶质粒转染至感染了VTT病毒的CV-1细胞中,培养3~5小时后更换一次培养基;
步骤1.4,将步骤1.3得到的CV-1细胞在培养箱继续培养48小时后,收集CV-1细胞,反复冻融三次,进行超声破碎,获得病毒悬液,并将其保存于-80℃的温度下。
步骤2具体按照下述步骤进行:
步骤2.1,在6孔板中培养CV-1细胞,至70~80%覆盖;
步骤2.2,将步骤1收集的病毒悬液,做10倍倍比稀释后感染CV-1细胞,在37℃培养2~3小时;
步骤2.3,在步骤2.2处理后的六孔板的每孔覆盖2~3ml琼脂DMEM培养基,培养2~3天;
步骤2.4,将经过步骤2.3处理后的6孔板在荧光显微镜下观察绿色荧光蛋白表达,挑取孤立的有绿色荧光的病毒噬斑进行连续单斑纯化,直至病毒纯化,得到去除VGF基因的重组天坛株溶瘤痘苗病毒。
去除VGF基因的重组天坛株溶瘤痘苗病毒作为疫苗、生物载体和抗肿瘤药物的应用。
本发明的有益效果是
本发明去除VGF基因的重组天坛株溶瘤痘苗病毒VTT-VGFE,降低了天坛株溶瘤痘苗病毒的毒性,提高了天坛株溶瘤痘苗病毒的肿瘤选择性,为痘苗病毒作为载体研发活疫苗及肿瘤生物治疗提供了良好工具,能够适用于治疗肺癌、肝癌、黑色素瘤等肿瘤。
本发明去除VGF基因的重组天坛株溶瘤痘苗病毒VTT-VGFE能够实现天坛株溶瘤痘苗病毒颗粒在体内分布的实时监测功能,可用于在活体水平上研究病毒与宿主细胞的作用。
附图说明
图1是本发明去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法中打靶质粒pMV-VTTVGFE的图谱;
图2是本发明产生的噬斑图,图2A为去除VGF基因的重组天坛株溶瘤痘苗病毒在明场下的噬斑图;图2B为本发明去除VGF基因的重组天坛株溶瘤痘苗病毒在暗场下的绿色荧光噬斑图;
图3是本发明去除VGF基因的重组天坛株溶瘤痘苗病毒的VGF区PCR鉴定图;
图4是本发明去除VGF基因的重组天坛株溶瘤痘苗病毒毒性试验中1×105PFU/只/200μl剂量下的小鼠的生存曲线图;
图5是本发明去除VGF基因的重组天坛株溶瘤痘苗病毒毒性试验中1×104PFU/只/200μl剂量下的小鼠的生存曲线图;
图6是本发明去除VGF基因的重组天坛株溶瘤痘苗病毒对肿瘤细胞的杀伤力实验中,VTT-VGFE和VTT对肺癌细胞A549的杀伤力情况对比图;
图7是本发明去除VGF基因的重组天坛株溶瘤痘苗病毒对肿瘤细胞的杀伤力实验中,VTT-VGFE病毒和VTT对肝癌细胞HepG2的杀伤力情况对比图。
具体实施方式
下面结合附图以及具体实施方式对本发明进行详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所使用的材料、试剂,如无特殊说明,均可从商业途径获得。
本发明中野生痘苗病毒天坛株的DNA序列为GenBank号为AF095689.1的序列(VRL14-FEB-2000)。
以下实施例中的野生型痘苗病毒天坛株在西安医学院分子病毒与病毒免疫学实验室(本实验室,下同)保藏。pMV-VTTVGFE质粒由本实验室保藏;细胞系CV-1、A549、HepG2购自中国医学科学院基础医学研究所基础医学细胞中心。
主要试剂和材料:
DMEM、胎牛血清购自hyclone公司,脂质体lipofectamine 2000转染试剂购自Invitrogen公司,病毒基因组提取试剂盒购自Biomiga公司,2×PCR Mix试剂和质粒提取试剂盒购自天根生化科技公司,Balb/c Nude小鼠购自北京维通利华实验动物技术有限公司。
一,去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法,具体按照下述步骤进行:
步骤1,将针对VTT病毒VGF基因的打靶质粒与VTT病毒在细胞内重组后,收集病毒:
步骤1.1,在T25细胞培养瓶中培养CV-1单层细胞,至70~80%覆盖;
步骤1.2,用VTT病毒感染CV-1细胞,在37℃的温度下培养2~3小时;
步骤1.3,将针对VTT病毒VGF基因的打靶质粒转染至感染了VTT病毒的CV-1细胞,培养3~5小时后更换一次培养基;步骤1.4,将步骤1.3得到的CV-1细胞在培养箱继续培养48小时后,收集CV-1细胞,反复冻融三次,进行超声破碎,获得病毒悬液,并将其保存于-80℃的温度下。
其中,针对VTT病毒VGF基因的打靶质粒为pMV-VTTVGFE,如图1所示,pMV-VTTVGFE含有出发载体pMV-Pro、痘苗病毒天坛株VGF基因的上游同源重组臂VTT-VGFL和下游同源重组臂VTT-VGFR,在VTT-VGFL与VTT-VGFR之间有荧光标记基因EGFP和1个多克隆位点,其中荧光标记基因EGFP由启动子P7.5控制表达,在多克隆位点处包含启动子P11。
步骤2,筛选纯化重组病毒,得到去除VGF基因的重组天坛株溶瘤痘苗病毒:
步骤2.1,在6孔板中培养CV-1细胞,至70~80%覆盖;
步骤2.2,将步骤1收集的病毒悬液,做10倍倍比稀释后感染CV-1细胞,并37℃培养2~3小时;
步骤2.3,在步骤2.2处理后的六孔板的每孔覆盖2~3ml琼脂DMEM培养基,培养2~3天;
步骤2.4,将经过步骤2.3处理后的6孔板在荧光显微镜下观察绿色荧光蛋白表达,挑取如图2b所示的孤立的有绿色荧光的病毒噬斑进行连续单斑纯化,直至病毒纯化,得到去除VGF基因的重组天坛株溶瘤痘苗病毒。
其中,图2a为重组病毒VTT-VGFE在明场下的噬斑图,本色为灰褐色,但是为了满足专利法对专利附图的要求,将其灰度,使其丧失了原本的颜色属性;
图2b为重组病毒VTT-VGFE在暗场下的绿色荧光噬斑图,本色为荧光绿色,但是为了满足专利法对专利附图的要求,将其灰度,使其丧失了原本的颜色属性;
步骤3,鉴定步骤2到的重组天坛株溶瘤痘苗病毒VTT-VGFE
步骤3.1,将步骤2得到的病毒在CV-1细胞内进行小量增殖,收集病毒,病毒悬液经超声处理后分两份,其中一份贮存在-80℃,另一份按照病毒DNA提取试剂盒说明书提取重组病毒VTT-VGFE的DNA,同样提取VTT病毒的DNA;
步骤3.2,对重组病毒VTT-VGFE进行PCR鉴定
VGF区鉴定引物:
JDVGF-F:TATAGGTTCCGTAAGCAAAG
JDVGF-R:TGATACGGAACCACCCACTG
以上述提取的重组痘苗病毒的DNA为模板,用上述鉴定引物进行PCR反应,同时设置野生型VTT为阴性对照。
PCR反应体系:
PCR反应条件按天根PCR试剂说明书进行,退火温度为60℃。
PCR结果:VTT的VGF区片段为991bp,重组病毒VTT-VGFE的VGF区替换片段为1533bp。PCR产物经1%琼脂糖凝胶电泳,结果如图3所示,M为markerIII,1和2为重组病毒VTT-VGFE的1号和2号,只包含1533bp片段;3为阳性对照即打靶质粒pMV-VTTVGFE,条带为1533bp;4为阴性对照即VTT,条带为991bp。结果表明重组病毒VTT-VGFE的VGF基因已被去除,且该病毒完全不包含野生VTT,重组病毒VTT-VGFE已完全纯化。
步骤3.3,对VTT-VGFE病毒DNA进行VGF区测序,结果表明VGF基因被敲除,被敲除的DNA序列见序列1,外源基因EGFP已***VGF区段,***的DNA序列见序列2。以上结果表明,重组病毒VTT-VGFE构建成功。
二,VTT-VGFE病毒毒性实验
取两组6-8周龄雌性Balb/c Nude小鼠,每组5只,腹腔接种病毒,VTT-VGFE为实验组,VTT为对照组,接种剂量为1×105PFU/只/200μl或1×104PFU/只/200μl,之后每天观察动物的存活率及存活时间。结果如图4和图5所示,相同剂量下,与VTT组相比,VTT-VGFE组小鼠存活时间显著延长;在低剂量组,VTT-VGFE组的小鼠存活率为60%,显著高于VTT组。结果表明,VTT-VGFE半数致死剂量显著提高,病毒毒性显著降低,去除VGF基因极大的提高了溶瘤痘苗病毒的安全性。
三,病毒对肿瘤细胞的杀伤力实验
肺癌细胞A549和肝癌细胞HepG2分别铺于96孔板内,每孔1×105个细胞,A549或HepG2细胞分别感染10MOI、1MOI和0.1MOI的VTT-VGFE病毒和10MOI、1MOI和0.1MOI的VTT病毒,每种病毒每剂量重复3孔,VTT-VGFE为实验组,VTT为对照组,72小时后通过CCK-8评估病毒对细胞的杀伤能力。结果如图6和图7显示,相同感染系数下,VTT-VGFE对A549或HepG2的杀伤能力与VTT一致,差异无统计学意义,说明去除VGF基因不影响病毒对肿瘤细胞的杀伤能力,VTT-VGFE可高效杀伤肿瘤细胞。
以上结果表明,本发明提供了一种去除VGF基因的减毒天坛株溶瘤痘苗病毒VTT-VGFE,该病毒显著降低了VTT病毒毒性,但不影响病毒对肿瘤细胞的杀伤能力,本发明可用于研发活疫苗,并可为临床提供抗肿瘤药物。
<110>西安彤盛生物科技有限公司
<120>去除VGF基因的重组天坛株溶瘤痘苗病毒及制备和应用
<130>无
<210>1
<211>364
<212>DNA
<213>VGF区敲除的DNA序列
<400>1
CTATGATAATCAGATCATTCGCCGATAGTGGTAACGCTATCGAAACGACATTGCCAGAAA 60
TTACAAACGCTACAACAGATATTCCAGCTATCAGATTATGCGGTCCAGAGGGAGATGGAT 120
ATTGTTTACACGGTGACTGTATCCACGCTAGAGATATTGACGGTATGTATTGTAGATGCT 180
CTCATGGTTATACAGGCATTAGATGTCAGCATGTAGTATTAGTAGACTATCAACGTTCAG 240
AAAAACCAAACACTACAACGTCATATATCCCATCTCCCGGTATTATGCTTGTATTAGTAG 300
GCATTATTATTATTACGTGTTGTCTATTATCTGTTTATAGGTTCACTCGACGAACTAAAC 360
TACC 364
<210>2
<211>1085
<212>DNA
<213>VGF区***的DNA序列
<400>2
AAGCTTCAACTGTCCATGGGCCCGCGGCCGCTCGAGCATCTGGTAATTTATAGCATAGAA 60
AAAAACAAAATGAAATTCTACTATATTTTTACATACATATATTCTAAATATGAAAGTGGT 120
GATTGTGACTAGCGTAGCATCGCTCATCTATATACTATATAGTAATACCAATAGAGCTCA 180
AGACTACGACTAGTAAACTGATACAATCTCTTATATGTGGGTAATGTTCTCGATGTCGAT 240
AGCCATATGCCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCACCCGC 300
TTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATGGAATTCATGGTGAGCAAG 360
GGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC 420
GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC 480
CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACC 540
CTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC 600
TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGAC 660
GGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC 720
GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTAC 780
AACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG 840
AACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG 900
CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC 960
CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC 1020
GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGATCTGGATCC 1080
CCGGG
Claims (8)
1.去除VGF基因的重组天坛株溶瘤痘苗病毒,其特征在于,去除天坛株溶瘤痘苗病毒VGF基因,其中VGF基因的碱基序列如序列表1所示。
2.根据权利要求1所述的去除VGF基因的重组天坛株溶瘤痘苗病毒,其特征在于,所述重组天坛株溶瘤痘苗病毒中***了绿色荧光蛋白基因EGFP,所述绿色荧光蛋白基因EGFP由启动子P7.5控制表达,所述绿色荧光蛋白基因EGFP位于重组天坛株溶瘤痘苗病毒的VGF区,所述绿色荧光蛋白基因EGFP的碱基序列如序列表2所示。
3.根据权利要求1所述的去除VGF基因的重组天坛株溶瘤痘苗病毒,其特征在于,所述重组天坛株溶瘤痘苗病毒的出发病毒为天坛株。
4.去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法,其特征在于,具体按照下述步骤进行:
步骤1,将针对VTT病毒VGF基因的打靶质粒与VTT病毒在细胞内重组后,收集病毒;
步骤2,筛选纯化重组病毒,得到去除VGF基因的重组天坛株溶瘤痘苗病毒。
5.根据权利要求4所述的去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法,其特征在于,所述步骤1中针对VTT病毒VGF基因的打靶质粒为pMV-VTTVGFE,所述打靶质粒pMV-VTTVGFE含有出发载体pMV-Pro、痘苗病毒天坛株VGF基因的上游同源重组臂VTT-VGFL和下游同源重组臂VTT-VGFR,在VTT-VGFL与VTT-VGFR之间有荧光标记基因EGFP和1个多克隆位点,其中荧光标记基因EGFP由启动子P7.5控制表达,多克隆位点处包含启动子P11。
6.根据权利要求4的去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法,其特征在于,所述步骤1具体按照下述步骤进行:
步骤1.1,在T25细胞培养瓶中培养CV-1单层细胞,至70~80%覆盖;
步骤1.2,用VTT病毒感染CV-1细胞,在37℃的温度下培养2~3小时;
步骤1.3,将针对VTT病毒VGF基因的打靶质粒转染至感染了VTT病毒的CV-1细胞,培养3~5小时后,更换一次培养基;
步骤1.4,将步骤1.3得到的CV-1细胞在培养箱继续培养48小时后,收集CV-1细胞,反复冻融三次,进行超声破碎,获得病毒悬液,并将其保存于-80℃的温度下。
7.根据权利要求4述的去除VGF基因的重组天坛株溶瘤痘苗病毒的制备方法,其特征在于,所述步骤2具体按照下述步骤进行:
步骤2.1,在6孔板中培养CV-1细胞,至70~80%覆盖;
步骤2.2,将步骤1收集的病毒悬液,做10倍倍比稀释后感染CV-1细胞,在37℃培养2~3小时;
步骤2.3,在步骤2.2处理后的六孔板的每孔覆盖2~3ml琼脂DMEM培养基,培养2~3天;
步骤2.4,将经过步骤2.3处理后的6孔板在荧光显微镜下观察绿色荧光蛋白表达,挑取孤立的有绿色荧光的病毒噬斑进行连续单斑纯化,直至病毒纯化,得到去除VGF基因的重组天坛株溶瘤痘苗病毒。
8.去除VGF基因的重组天坛株溶瘤痘苗病毒作为疫苗、生物载体和抗肿瘤药物的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910012884.5A CN110205307A (zh) | 2019-01-07 | 2019-01-07 | 去除vgf基因的重组天坛株溶瘤痘苗病毒及制备和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910012884.5A CN110205307A (zh) | 2019-01-07 | 2019-01-07 | 去除vgf基因的重组天坛株溶瘤痘苗病毒及制备和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110205307A true CN110205307A (zh) | 2019-09-06 |
Family
ID=67785003
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910012884.5A Pending CN110205307A (zh) | 2019-01-07 | 2019-01-07 | 去除vgf基因的重组天坛株溶瘤痘苗病毒及制备和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110205307A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109750056A (zh) * | 2019-01-07 | 2019-05-14 | 西安医学院 | 去除天坛株vgf基因的打靶质粒及其制备方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105121635A (zh) * | 2013-04-10 | 2015-12-02 | 东源生物医药科技(上海)有限公司 | 突变型痘苗病毒株、其用途及其制造方法 |
CN107604004A (zh) * | 2017-09-22 | 2018-01-19 | 西安医学院 | 用于痘苗病毒天坛株tk基因的示踪打靶质粒及其制备方法 |
CN109477089A (zh) * | 2016-05-30 | 2019-03-15 | 安斯泰来制药株式会社 | 新型基因重组牛痘病毒 |
-
2019
- 2019-01-07 CN CN201910012884.5A patent/CN110205307A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105121635A (zh) * | 2013-04-10 | 2015-12-02 | 东源生物医药科技(上海)有限公司 | 突变型痘苗病毒株、其用途及其制造方法 |
CN109477089A (zh) * | 2016-05-30 | 2019-03-15 | 安斯泰来制药株式会社 | 新型基因重组牛痘病毒 |
CN107604004A (zh) * | 2017-09-22 | 2018-01-19 | 西安医学院 | 用于痘苗病毒天坛株tk基因的示踪打靶质粒及其制备方法 |
Non-Patent Citations (2)
Title |
---|
J. ANDREA MCCART,等: "Systemic Cancer Therapy with a Tumor-selective Vaccinia Virus Mutant Lacking Thymidine Kinase and Vaccinia Growth Factor Genes" * |
R. MARK L. BULLER,等: "Deletion of the Vaccinia Virus Growth Factor Gene Reduces Virus Virulence" * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109750056A (zh) * | 2019-01-07 | 2019-05-14 | 西安医学院 | 去除天坛株vgf基因的打靶质粒及其制备方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109810953A (zh) | 去除tk基因的重组天坛株溶瘤痘苗病毒及其制备和应用 | |
RU2581910C2 (ru) | Процесс получения поксвирусов и композиции поксвирусов | |
RU2002124622A (ru) | Измененный штамм модифицированного вируса коровьей оспы ankara (mva) | |
CN109554353A (zh) | 分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途 | |
CN110218706B (zh) | 表达h7n9亚型高致病性禽流感病毒ha蛋白的重组火鸡疱疹病毒的构建与应用 | |
CN102732545B (zh) | 鸭瘟病毒转移载体pUC-ΔgC-EGFP及gC缺失重组株DPV-ΔgC-EGFP | |
MX2012000979A (es) | Composición enzimática para la digestión de embriones de pollo. | |
CN108913813A (zh) | 用于鉴别DAdV-2和DAdV-3的引物组 | |
CN109321535A (zh) | 一种热稳定的新城疫病毒弱毒疫苗候选株 | |
CN108588026A (zh) | 高表达il10的临床级间充质干细胞的制备方法及其用途 | |
CN1800374A (zh) | 一种重组伪狂犬病-猪繁殖与呼吸综合征基因工程毒株及应用 | |
CN109536529A (zh) | 一种高效重组痘苗病毒载体及其建立方法 | |
CN113201508A (zh) | 一种重组新城疫溶瘤病毒及制备方法与应用 | |
CN110205308A (zh) | 一种表达ha基因的重组火鸡疱疹病毒及其应用 | |
CN110205307A (zh) | 去除vgf基因的重组天坛株溶瘤痘苗病毒及制备和应用 | |
CN108866240A (zh) | 用于鉴别DAdV-3和DAdV-A的引物和酶及其应用 | |
Wang et al. | Establishment and characterization of duck embryo epithelial (DEE) cell line and its use as a new approach toward DHAV-1 propagation and vaccine development | |
CN110205304A (zh) | 一种重组天坛株溶瘤痘苗病毒及制备方法和应用 | |
CN113226336A (zh) | 一种在细胞中递送基因的方法 | |
Boyle | Genus avipoxvirus | |
CN113388586B (zh) | 一种溶瘤病毒ndv-nrp1及其构建方法与应用 | |
CN114657154B (zh) | 一种羊传染性脓疱病毒减毒株的制备方法及其应用 | |
CN108842000A (zh) | 用于鉴别DAdV-3和DAdV-A的引物组 | |
RU2482129C2 (ru) | Штамм вируса болезни ньюкасла для создания на его основе кандидатного противоракового препарата и изучения механизмов онколизиса | |
CN103589693B (zh) | 一种表达ibdv vp2和法氏囊素三肽嵌合蛋白重组火鸡疱疹病毒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190906 |