CN113481241A - Recombinant vaccinia virus vector with H3L gene knocked out - Google Patents
Recombinant vaccinia virus vector with H3L gene knocked out Download PDFInfo
- Publication number
- CN113481241A CN113481241A CN202110679461.6A CN202110679461A CN113481241A CN 113481241 A CN113481241 A CN 113481241A CN 202110679461 A CN202110679461 A CN 202110679461A CN 113481241 A CN113481241 A CN 113481241A
- Authority
- CN
- China
- Prior art keywords
- vaccinia virus
- gene
- vector
- nucleic acid
- homologous recombination
- 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
- 241000700618 Vaccinia virus Species 0.000 title claims abstract description 140
- 101150082093 H3L gene Proteins 0.000 title claims abstract description 111
- 239000013598 vector Substances 0.000 title claims abstract description 108
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 105
- 230000006801 homologous recombination Effects 0.000 claims abstract description 76
- 238000002744 homologous recombination Methods 0.000 claims abstract description 76
- 102200082900 rs33983205 Human genes 0.000 claims abstract description 67
- 150000007523 nucleic acids Chemical group 0.000 claims description 61
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 52
- 239000013605 shuttle vector Substances 0.000 claims description 51
- 108700008625 Reporter Genes Proteins 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 14
- 238000003209 gene knockout Methods 0.000 claims description 13
- 238000000746 purification Methods 0.000 claims description 9
- 230000003362 replicative effect Effects 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 5
- 241000700605 Viruses Species 0.000 abstract description 32
- 230000003472 neutralizing effect Effects 0.000 abstract description 16
- 229960005486 vaccine Drugs 0.000 abstract description 11
- 238000001727 in vivo Methods 0.000 abstract description 8
- 206010028980 Neoplasm Diseases 0.000 abstract description 7
- 206010046865 Vaccinia virus infection Diseases 0.000 abstract description 7
- 208000007089 vaccinia Diseases 0.000 abstract description 7
- 238000001415 gene therapy Methods 0.000 abstract description 5
- 101900350956 Vaccinia virus Envelope protein H3 Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 34
- 239000005090 green fluorescent protein Substances 0.000 description 21
- 239000000427 antigen Substances 0.000 description 18
- 108091007433 antigens Proteins 0.000 description 18
- 102000036639 antigens Human genes 0.000 description 18
- 210000003501 vero cell Anatomy 0.000 description 14
- 208000015181 infectious disease Diseases 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 12
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 8
- 108091006047 fluorescent proteins Proteins 0.000 description 8
- 102000034287 fluorescent proteins Human genes 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 8
- 241000700649 Vaccinia virus Tian Tan Species 0.000 description 7
- 230000002163 immunogen Effects 0.000 description 7
- 238000012423 maintenance Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 229930193140 Neomycin Natural products 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 238000010353 genetic engineering Methods 0.000 description 6
- 229960004927 neomycin Drugs 0.000 description 6
- 108700026220 vif Genes Proteins 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000012096 transfection reagent Substances 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 102000002464 Galactosidases Human genes 0.000 description 4
- 108010093031 Galactosidases Proteins 0.000 description 4
- 241000700647 Variola virus Species 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000013600 plasmid vector Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000013566 allergen Substances 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 244000045947 parasite Species 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 241000711798 Rabies lyssavirus Species 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 208000035415 Reinfection Diseases 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 108091005948 blue fluorescent proteins Proteins 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 229960004854 viral vaccine Drugs 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- 229940124718 AIDS vaccine Drugs 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000710842 Japanese encephalitis virus Species 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000700625 Poxviridae Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000002941 microtiter virus yield reduction assay Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- 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
- 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
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24122—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
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/50—Vectors for producing vectors
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of molecular biology and immunology, in particular to a recombinant vaccinia virus vector with a H3L gene knocked out; the recombinant vaccinia virus vector has an H3L homologous recombination arm sequence, and is used for constructing a vaccinia virus vector inserted with an exogenous gene in a vaccinia virus H3L gene region; the invention provides a recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibodies existing in vivo, wherein the H3L encoding gene of the recombinant vaccinia virus vector is knocked out or damaged and cannot normally express the H3L gene, and the vaccinia virus vector can be used as a tumor vaccine or a gene therapy vector and can be used for preventing or treating various tumors.
Description
Technical Field
The invention relates to the technical field of molecular biology and immunology, in particular to a recombinant vaccinia virus vector with a H3L gene knocked out.
Background
Vaccinia Virus (Vaccinia Virus), belonging to the genus Orthomyxovirus of the family Poxviridae, has been widely used worldwide as a vaccine for the prevention of smallpox infection, making smallpox the first malignant infectious disease to be completely eradicated by humans (Riva, G.et. al., 1979.A milestone in the history of infection. eradiation of smallpox. MMW Munch Meachhenschr, 121(50), 1669-70). In the last two decades, with the rapid development of genetic engineering techniques, vaccinia viruses have been used in research to design gene expression vectors, or in the development of recombinant live virus vaccines (Moss, B.1996.genetic engineering viruses for recombinant gene expression, vaccination, and safety. ProcNatl Acad Sci USA 93(21), 11341-8; Paoletti, E.et al, 1996.Applications of pox viruses vectors to vaccination: an update. Proc Natl Acad Sci USA 93(21), 11349-53). Various recombinant vaccinia virus vaccines have been used successfully to express rabies virus, Japanese encephalitis virus, and other viral antigens and to control animal infections (Kieny, M.P. et al, 1984.expression of viruses from a recombinant vaccine virus. Nature312(5990), 163-6; Konishi, E.et al, 1992.A high expressed family-restricted vaccine strain, NYVAC, encoding the prM, E, and NS1genes of Japanese infectious virus precursors JEV virus strain 190(1), 454-8). There have also been many important advances in human recombinant live virus vaccines, including preliminary human immune observations including measles, EBV, HAV, HBV, HPV, HIV and rabies viruses (Guofei et al, 2001, The construction of expression vectors for non-replicating recombinant vaccinia virus Tiantan strain C-K deletion region and The study of biological properties of recombinant viruses. viral acta 17(1), 24-28; McMichael, A.et al, 2002.The request for AIDS vaccine: is The CD8+ T-cell ap pro plasmid Nat Rev Immunol 2(4), 283-91; Moss, B.1996.genetic engineering for gene expression vector expression, vaccination, and safety. Proc. Natl Acad Sci 93(21), 11341-8; ZJ.J.J.65. vaccine, see 141. 7. and 141. biological genes).
The vaccinia virus Tiantan strain is a peculiar vaccinia virus strain in China, has been used for a long time in a large number of people in China as a vaccine strain for preventing smallpox infection, has the characteristics of relatively weak toxicity and safe use, and is an ideal strain for developing a genetic engineering virus vector and a recombinant live vaccine suitable for China. The sequencing of the whole genome of the Chinese vaccinia virus Tiantan strain was completed in 1997, which provides important background data for the research and elucidation of the unique biological characteristics of the vaccinia virus Tiantan strain and the genetic engineering modification (King et al, 1997, analysis of the whole genome structural characteristics of the vaccinia virus Tiantan strain, China science (C edition) 27(6), 562 and 567).
With the development of molecular biology, some viruses have been widely researched and developed as recombinant viral vectors due to their characteristics of good safety, strong immunogenicity, and convenience for gene manipulation. At present, the method is mainly applied to the medical fields of vaccines, gene therapy and the like. The poxvirus vector is large, the genome of the poxvirus vector is about 180k, and the poxvirus vector can accommodate large exogenous gene segments, so that the poxvirus vector can be used as an excellent viral vector for mediating the delivery of target proteins or coding genes thereof in vivo.
However, vaccinia virus, as a recombinant viral vector, has a strong vector response that can be activated to produce a strong immune response against vaccinia virus itself, including a strong neutralizing antibody response. Moreover, the response is long lasting and the neutralizing antibody response is provided for the lifetime of the individual vaccinated with the vaccinia virus vector. Thus, the presence of high levels of neutralizing antibodies in vivo will greatly affect reinfection with the recombinant vaccinia virus vector of interest, thereby affecting the effectiveness of viral vector drug delivery. This feature also limits the repeated or multiple applications of vaccinia virus vectors.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a recombinant vaccinia virus vector for knocking out H3L gene, which selects main neutralizing antigen of vaccinia virus as a target spot, and researches whether knocking out or destroying the target spot can escape from the existing neutralizing antibody in vivo, thereby increasing the use frequency of the recombinant vaccinia virus vector in vivo.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention relates to a gene knockout or a knockout technology, which is a genetic engineering technology and refers to a technology or a method for replacing an endogenous normal homologous gene by using an exogenous mutated gene through a homologous recombination method, so that the endogenous gene is inactivated to express the character of a mutant. In the invention, the H3L knockout is that a mutation is introduced into the H3L gene, and the endogenous normal H3L gene of the vaccinia virus is replaced by means of homologous recombination, so that the H3L gene cannot be normally expressed.
The "Recombinant virus vector" or "Recombinant virus" as referred to herein is a vector produced by a DNA recombination technique and used for producing a viral vaccine or producing a gene therapy.
The term "Recombinant vaccinia virus vector" or "Recombinant vaccinia virus" as used herein refers to a vaccinia virus vector or vaccinia virus produced by Recombinant DNA techniques, and can be used for producing viral vaccines or vectors for gene therapy. In the present invention, a "recombinant vaccinia virus vector" or "recombinant vaccinia virus" is produced by homologous recombination of a shuttle vector having a homologous arm sequence of a target gene with vaccinia virus in a target cell, and then purified by selection of a reporter gene or a resistance gene carried by the recombinant vaccinia virus.
The term "shuttle plasmid vector" or "shuttle vector" as used herein refers to an intermediate plasmid vector for introducing a gene sequence of interest into the genome of another species. In the present invention, the "shuttle plasmid vector" or "shuttle vector" refers to an intermediate plasmid vector for introducing a target gene sequence into a vaccinia virus vector by homologous recombination, characterized in that the shuttle vector contains two homologous recombination arms having the same sequence as a partial sequence of a vaccinia virus genome.
The term "homologous recombination arm" or "homology arm" as used herein refers to a nucleic acid sequence that is identical to a vaccinia virus endogenous gene.
A recombinant vaccinia virus vector with a H3L gene knocked out, wherein a gene encoding H3L of the recombinant vaccinia virus vector is knocked out, and the H3L gene cannot be normally expressed.
The invention is further configured to: the vaccinia virus vector is a replicative vaccinia virus vector or a non-replicative vaccinia virus vector.
The invention is further configured to: the H3L knockout refers to that the endogenous normal H3L gene of the vaccinia virus is replaced by homologous recombination through introducing mutation in the H3L gene, so that the H3L gene cannot be normally expressed.
The invention is further configured to: the H3L knockout is performed by mutating the H3L gene by inserting a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene into the nucleic acid sequence at both ends of H3L, and further comprising one or more foreign genes other than the reporter gene or the resistance gene.
The invention is further configured to: the vaccinia virus promoter element is P7.5 or PE/L.
The invention is further configured to: the reporter gene is a fluorescent protein coding gene or a galactosidase coding gene.
The invention is further configured to: the resistance gene is a neomycin encoding gene, and when the resistance gene is used for purifying a recombinant vaccinia virus vector, selective pressure is applied.
The invention is further configured to: the exogenous gene is a non-vaccinia virus-derived gene other than the reporter gene or the resistance gene.
The invention is further configured to: the nucleic acid sequences at two ends of the H3L are respectively an H3L-L homologous recombination arm and an H3L-R homologous recombination arm.
The invention is further configured to: the H3L knockout recombinant vaccinia virus vector further has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence.
The invention is further configured to: the H3L knockout recombinant vaccinia virus vector is rvv-delta H3L/G.
The invention is further configured to: the recombinant vaccinia virus vector capable of escaping the existing anti-vaccinia virus neutralizing antibody existing in vivo is a recombinant vaccinia virus vector with an H3L gene knockout.
The invention is further configured to: the H3L gene knockout recombinant vaccinia virus vector contains an H3L-L homologous recombination arm sequence, and the H3L gene knockout recombinant vaccinia virus vector also contains an H3L-R homologous recombination arm sequence.
A shuttle vector for constructing a recombinant vaccinia virus vector with an H3L gene knockout, wherein the shuttle vector comprises an H3L homologous recombination arm sequence.
The invention is further configured to: the shuttle vector contains H3L-L homologous recombination arm sequences.
The invention is further configured to: the shuttle vector also contains the H3L-R homologous recombination arm sequence.
The invention is further configured to: the shuttle vector also inserts a nucleic acid sequence between the H3L homologous recombination arm sequences, wherein the nucleic acid sequence comprises at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, and the nucleic acid sequence also comprises one or more exogenous genes besides the reporter gene or the resistance gene.
The invention is further configured to: the vaccinia virus promoter element is P7.5 or PE/L.
The invention is further configured to: the reporter gene is a fluorescent protein coding gene or a galactosidase coding gene.
The invention is further configured to: the resistance gene is a neomycin encoding gene, and when the resistance gene is used for purifying a recombinant vaccinia virus vector, selective pressure is applied.
The invention is further configured to: the exogenous gene is a non-vaccinia virus-derived gene other than the reporter gene or the resistance gene.
The invention is further configured to: the nucleic acid sequences at two ends of the H3L are respectively an H3L-L homologous recombination arm and an H3L-R homologous recombination arm.
The invention is further configured to: the shuttle vector also has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence.
The invention is further configured to: the shuttle vector is p.DELTA.H 3L-GFP.
A preparation method for constructing a recombinant vaccinia virus vector with an H3L gene knockout function comprises the following steps:
s1, designing primers, and amplifying two homologous recombination arms of H3L by PCR respectively;
s2, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the H3L homologous recombination arm sequence;
s3, transfecting the shuttle vector into a vaccinia virus infected cell;
s4, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on the new cells.
Advantageous effects
Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:
the invention selects the main neutralizing antigen of the vaccinia virus as a target spot, and researches whether knocking out or destroying the target spot can escape the existing neutralizing antibody in vivo, thereby increasing the using times of the recombinant vaccinia vector in vivo, knocking out or destroying the H3L encoding gene of the recombinant vaccinia vector, and not normally expressing the H3L gene, and the vaccinia virus vector can be used as a tumor vaccine or a gene therapy vector and can be used for preventing or treating various tumors.
Drawings
FIG. 1 is a plasmid map of shuttle vector p.DELTA.H3L-GFP with H3L-L and H3L-R homologous recombination arm sequences;
FIG. 2 is a diagram showing the double restriction enzyme identification of the shuttle vector p.DELTA.H3L-GFP with H3L-L and H3L-R homologous recombination arm sequences;
FIG. 3 is a diagram showing the identification of recombinant vaccinia virus vector rvv- Δ H3L/G by PCR amplification;
FIG. 4 shows an expression identification chart of recombinant vaccinia virus vector rvv- Δ H3L/G;
FIG. 5 shows the results of detection of neutralizing antibodies in example 5.
FIG. 6 shows the results of the kinetics of viral replication in example 6, including the viral dose in infected cells and the viral dose in the infection supernatant.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention will be further described with reference to the following examples.
Referring to FIGS. 1-6: the invention aims to provide a recombinant vaccinia virus vector with a H3L gene knocked out, wherein a H3L encoding gene of the recombinant vaccinia virus vector is knocked out, and the H3L gene cannot be normally expressed.
In one embodiment of the invention, the Vaccinia virus vector is a replicating Vaccinia virus vector, such as a Vaccinia virus Tiantan strain, e.g., strain 752-1, or a non-replicating Vaccinia virus vector, such as a Vaccinia virus attenuated vaccine Ankara strain (Modified Vaccinia Ankara, MVA).
In one embodiment of the invention, H3L knockout refers to replacement of the endogenous normal H3L gene of vaccinia virus by homologous recombination by introducing a mutation inside the H3L gene, such that the H3L gene is not normally expressed.
In one embodiment of the invention, the H3L knockout is performed by mutating the H3L gene by inserting a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene into the nucleic acid sequence at both ends of H3L, the nucleic acid sequence further comprising one or more exogenous genes other than the reporter gene or the resistance gene.
In one embodiment of the invention, the vaccinia virus promoter element is P7.5 or PE/L, preferably, the nucleic acid sequence of P7.5 is shown as SEQ ID NO. 13 and the nucleic acid sequence of PE/L is shown as SEQ ID NO. 14.
In one embodiment of the present invention, the reporter gene is a fluorescent protein coding gene or a galactosidase coding gene (Lac Z), preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene, a yellow fluorescent protein coding gene, a red fluorescent protein coding gene or a blue fluorescent protein coding gene, more preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene (EGFP), and the nucleic acid coding sequence thereof is shown in SEQ ID NO. 15.
In one embodiment of the invention, the resistance gene is a neomycin encoding gene and selective pressure is applied when the resistance gene is used in the purification of recombinant vaccinia virus vectors.
In one embodiment of the present invention, the foreign gene is a gene derived from non-vaccinia virus itself except for a reporter gene or a resistance gene, and the target antigen or immunogen-encoding gene selected according to the purpose of use may be any one or more immunogens of interest, preferably, the target immunogen is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite or other intact microorganism, and further, the target antigen or immunogen is a tumor antigen or an infection-associated antigen.
In one embodiment of the invention, the nucleic acid sequences at the two ends of H3L are respectively H3L-L homologous recombination arm and H3L-R homologous recombination arm, preferably, the nucleic acid sequence of the H3L-L homologous recombination arm is shown as SEQ ID NO. 1, and the nucleic acid sequence of the H3L-R homologous recombination arm is shown as SEQ ID NO. 2.
In one embodiment of the invention, the H3L knockout recombinant vaccinia virus vector further comprises a nucleic acid sequence as shown in SEQ ID NO. 16 inserted into the H3L homologous recombination arm sequence.
In one embodiment of the invention, the H3L knock-out recombinant vaccinia virus vector is rvv- Δ H3L/G.
In one embodiment of the invention, the recombinant vaccinia virus vector from which the existing anti-vaccinia virus neutralizing antibody can escape is a recombinant vaccinia virus vector with an H3L gene knockout.
In one embodiment of the invention, the H3L gene knockout recombinant vaccinia virus vector comprises an H3L-L homologous recombination arm sequence, and the H3L gene knockout recombinant vaccinia virus vector further comprises an H3L-R homologous recombination arm sequence, preferably, the H3L-L homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 1, and the H3L-R homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 2.
The invention also provides a shuttle vector for constructing the H3L gene knockout recombinant vaccinia virus vector, wherein the shuttle vector contains the H3L homologous recombination arm sequence.
In one embodiment of the invention, the shuttle vector comprises the H3L-L homologous recombination arm sequence, preferably the H3L-L homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 1.
In one embodiment of the invention, the shuttle vector further comprises an H3L-R homologous recombination arm sequence, preferably, the H3L-R homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further comprises a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, and one or more foreign genes other than the reporter gene or the resistance gene, inserted between the H3L homologous recombination arm sequences.
In one embodiment of the invention, the vaccinia virus promoter element is P7.5 or PE/L, the nucleic acid sequence of P7.5 is shown as SEQ ID NO. 13, and the nucleic acid sequence of PE/L is shown as SEQ ID NO. 14.
In one embodiment of the present invention, the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene (Lac Z), preferably, the fluorescent protein-encoding gene is a green fluorescent protein-encoding gene, a yellow fluorescent protein-encoding gene, a red fluorescent protein-encoding gene or a blue fluorescent protein-encoding gene, more preferably, the fluorescent protein-encoding gene is a green fluorescent protein-Encoding Gene (EGFP), and the nucleic acid-encoding sequence thereof is shown in SEQ ID NO. 15.
In one embodiment of the invention, the resistance gene is a neomycin encoding gene and selective pressure is applied when the resistance gene is used in the purification of recombinant vaccinia virus vectors.
In one embodiment of the present invention, the foreign gene is a gene derived from non-vaccinia virus itself except for a reporter gene or a resistance gene, and the target antigen or immunogen-encoding gene selected according to the purpose of use may be any one or more immunogens of interest, preferably, the target immunogen is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite or other intact microorganism, and further, the target antigen or immunogen is a tumor antigen or an infection-associated antigen.
In one embodiment of the invention, the nucleic acid sequences at the two ends of H3L are respectively H3L-L homologous recombination arm and H3L-R homologous recombination arm, preferably, the sequence of the H3L-L homologous recombination arm is shown as SEQ ID NO. 1, and the sequence of the H3L-R homologous recombination arm is shown as SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence, and the nucleic acid sequence is shown in SEQ ID NO. 16.
In one embodiment of the present invention, the shuttle vector is p.DELTA.H 3L-GFP, the nucleic acid sequence of which is shown in SEQ ID NO: 17.
The invention also provides a preparation method for constructing the H3L gene knockout recombinant vaccinia virus vector, which comprises the following steps:
step one, designing primers, and amplifying two homologous recombination arms of H3L by PCR respectively.
And step two, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the H3L homologous recombination arm sequence.
And step three, transfecting the shuttle vector into the vaccinia virus infected cell.
And step four, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on the new cells.
Wherein, the primer is a primer sequence which can be combined with two ends of H3L, and is preferably SEQ ID NO. 3H3L-LC-F primer, SEQ ID NO. 4H3L-LC-R primer, SEQ ID NO. 5H3L-RC-F primer or SEQ ID NO. 6H3L-RC-R primer.
The shuttle vector is characterized by containing H3L homologous recombination arm sequences.
In one embodiment of the invention, the shuttle vector contains the H3L-L homologous recombination arm sequence. Preferably, the H3L-L homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 1.
In one embodiment of the invention, the shuttle vector further comprises an H3L-R homologous recombination arm sequence. Preferably, the H3L-R homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 2.
In one embodiment of the invention, the shuttle vector further comprises a nucleic acid sequence inserted between the sequences of the H3L homologous recombination arms. Preferably, the nucleic acid sequence comprises at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene. In embodiments of the invention, the nucleic acid sequence may also include one or more additional exogenous genes in addition to the reporter gene or the resistance gene.
In one embodiment, the vaccinia virus promoter element is P7.5 or PE/L. Preferably, the nucleic acid sequence of P7.5 is shown as SEQ ID NO. 13, and the nucleic acid sequence of PE/L is shown as SEQ ID NO. 14.
In one embodiment, the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene (Lac Z). Preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene, a yellow fluorescent protein coding gene, a red fluorescent protein coding gene or a blue fluorescent protein coding gene. More preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene (EGFP), and the nucleic acid coding sequence of the fluorescent protein coding gene is shown in SEQ ID NO. 15.
In the present invention, the resistance gene is, for example, a Neomycin (Neomycin) encoding gene, and can be used for applying selective pressure when purifying a recombinant vaccinia virus vector.
In the present invention, the other foreign gene is a gene derived from a non-vaccinia virus itself in addition to the reporter gene or the resistance gene. The target antigen or immunogen-encoding gene may be selected according to the purpose of use. The immunogen of interest is any one or more immunogens. Preferably, the immunogen of interest is a peptide, antigen, hapten, carbohydrate, protein, nucleic acid, allergen, virus or part of a virus, bacterium, parasite or other whole microorganism. In addition, the antigen or immunogen of interest is a tumor antigen or an infection-associated antigen.
In the invention, the nucleic acid sequences at two ends of H3L are respectively an H3L-L homologous recombination arm and an H3L-R homologous recombination arm. Preferably, the H3L-L homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 1. Preferably, the H3L-R homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence, the sequence is shown in SEQ ID NO. 16.
In one embodiment of the present invention, the shuttle vector is p.DELTA.H 3L-GFP, the nucleic acid sequence of which is shown in SEQ ID NO: 17.
Among them, the Vaccinia virus is preferably a replicative Vaccinia virus vector such as Vaccinia virus Tiantan strain, e.g., 752-1 strain, or a non-replicative Vaccinia virus vector such as Vaccinia virus attenuated vaccine Ankara strain (Modified Vaccinia Ankara, MVA).
The cells are eukaryotic cell lines, preferably mammalian cell lines, such as 293T cells, 143B cells, Vero cells or BHK-21 cell lines.
Transfection is a technique well known in the art, i.e., delivery of the plasmid DNA of interest into cells, and includes, but is not limited to, physical methods (e.g., electroporation, cell extrusion, nanoparticles, and magnetic transfer), or chemical methods (e.g., calcium phosphate transfection, lipofection, DEAE-dextran or polyethyleneimine transfection), preferably, transfection is polyethyleneimine transfection, such as Turbofect (Thermo Fisher Scientific, R0531) transfection reagent.
For a better description of the invention, see examples 1-6:
example 1 p.DELTA.H 3L-GFP shuttle vector construction.
H3L-L (SEQ ID NO:1) and H3L-R (SEQ ID NO:2) fragments were amplified respectively by designing primers (Table 1) on the genomic DNA (preparation method reference example 3) of vaccinia virus wild-type rvv-WT (provided by Beijing biologicals) on the sequence of the cloned genome of the Techn strain poxvirus TP5, wherein the H3L-LC-F and H3L-LC-R primer pairs were used to amplify the H3L-L fragment, and the H3L-RC-F and H3L-RC-R primer pairs were used to amplify the H3L-R fragment.
Table 1: primers in example 1
In step 1, an intermediate vector p delta H3L-L-GFP with H3L-L homologous recombination arms is constructed. First, a linearized vector of about 5kbp was PCR-amplified using a pSC65-GFP vector (obtained by replacing LacZ expressing galactosidase on the pSC65 vector with EGFP gene expressing green fluorescent protein, supplied by Soviken Korea Tokyo Biotech Co., Ltd.) modified from pSC65 shuttle vector (addge, cat # 30327) (plasmid map shown in FIG. 1) as a template, and TKL-F and TKL-R (Table 1) primers. The linearized vector was then subjected to homologous recombination with the H3L-L fragment using the EasyGeno Rapid cloning kit (Tiangen, VI201), methods referenced in the kit instructions. The vector p.DELTA.H 3L-L-GFP with the H3L-L homology arm sequence was then constructed using recombinant transformation methods well known in the art.
In step 2, a shuttle vector p.DELTA.H 3L-GFP was constructed. Using the intermediate vector p.DELTA.H 3L-L-GFP constructed in step 1 as a template, a linearized vector of about 5kbp was amplified using TKR-F and TKR-R primer set (Table 1). The linearized vector was then subjected to homologous recombination with the H3L-R fragment using the EasyGeno Rapid cloning kit (Tiangen, VI201), methods referenced in the kit instructions. Then, a vector p delta H3L-GFP (a plasmid map is shown in a figure 1) with H3L-L and H3L-R homologous arm sequences is constructed by a recombinant transformation method well known in the art, and is stored after being sequenced and identified correctly. The vector p.DELTA.H 3L-GFP (the digestion system is shown in Table 2) was identified by using restriction enzymes EcoR V and Kpn I, and the digestion verification map is shown in FIG. 2.
Table 2: plasmid p.DELTA.H 3L-GFP restriction enzyme identification system (restriction enzyme for 2 hours at 37 ℃ C.)
| Enzyme digestion system | Volume of |
| Plasmid p.DELTA.H 3L- |
4 μ L, about 1 μ g |
| EcoR V (Baoyuan, goods number 1042A) | 1μL |
| Kpn I (Baozi, cat No. 1068A) | 1μL |
| Enzyme digestion buffer solution | 1μL |
| ddH2O | Make up to 10 mu L |
Example 2 recombinant vaccinia virus vector rvv- Δ H3L/G was constructed.
Recombinant vaccinia virus vectors were obtained in 293T cells as follows. On day 1, 293T cells were plated in 6-well cell culture plates (JET, TCP-010-6Well, incubated overnight at 37 ℃ in a carbon dioxide cell incubator. The following day, at 0.05MOI (i.e., 5X 10)4PFU (plaque forming Unit)/well) was added vaccinia virus wild strain rvv-WT (supplied by Beijing biologicals) and incubated in a 37 ℃ carbon dioxide cell incubator for two hours during which the shuttle vector/transfection reagent complex was prepared. Wherein the shuttle vector was p.DELTA.H 3L-GFP obtained in example 1, the transfection reagent was Turbofect (Thermo Fisher Scientific,r0531), the transfection dosage and the compounding method can be seen in the specification of the transfection reagent. After completion of the complexing system, the 293T cell supernatant was changed to 2 mL/well DMEM maintenance medium containing 2% Fetal Bovine Serum (FBS), and then the shuttle vector/transfection reagent complex was added. 48 hours after transfection, supernatant was removed, cells were harvested and resuspended in 0.5mL maintenance medium, freeze-thawed three times repeatedly, and recombinant cell lysate was then inoculated into Vero cells (R: (R) (R))
CCL-81) and incubated at 37 ℃ for 1 to 2 days. During which cytopathic effects are observed and single plaque purification is performed when the appropriate number of viral plaques are present (less than 20 plaques/well).
Single-spot purification:
and (3) observing virus plaques expressing green fluorescence under a fluorescence microscope, and marking.
Remove the supernatant, pick several well-dispersed green fluorescent plaques per well, and transfer to centrifuge tubes containing 0.5mL of maintenance medium, respectively.
Shaking and mixing the centrifuge tube containing the virus, repeatedly freezing and thawing for three times (about 5 minutes at minus 80 ℃ in a refrigerator and about 2 minutes at room temperature), finally shaking and mixing the mixture, and freezing and storing the mixture at minus 80 ℃.
Vero cells (T) were prepared on 6-well cell culture plates (JET, TCP-010-
CCL-81), virus was then removed from the-80 ℃ freezer, seeded onto Vero cells in 10 μ L each, and incubated at 37 ℃ for 2 to 3 days. During which cytopathic effects are observed and the next round of single plaque purification is performed when the appropriate number of viral plaques are present (less than 20 plaques/well).
At least six rounds of single-spot purification were repeated until the purity reached 100%.
Example 3 identification of recombinant vaccinia virus vector rvv- Δ H3L/G.
The purified vaccinia virus vector rvv-. DELTA.H 3L/G was inoculated onto Vero cells (1X 10)7Cells) were observed after 48 hours, and when 80% or more of the cells were infected, the cells were collected, centrifuged at 1800g for 5 minutes, and the supernatant was removed. Then using the genomeDNA extraction kit (TRAN, EE101-02) for extracting vaccinia virus genome DNA, wherein the specific method refers to kit specification, and finally 100 μ L of genome DNA is obtained and frozen at-20 ℃.
The result of PCR amplification of poxvirus genomic DNA using primers at both ends of H3L gene is shown in FIG. 3, the target band with size of 567bp can be amplified by using primer pair H3L-JF and EGPV 62-R (Table 3) in recombinant vaccinia virus vector rvv- Δ H3L/G genomic DNA, the target band with size of 1910bp can be amplified by using primer pair P7.5-F and H3L-RC-R (Table 3), the PCR amplification product sample is sent to Jinzhi corporation for sequencing, the sequence result is in line with the expectation, which indicates that H3L fragment is successfully knocked out.
Table 3: primers for identification in example 3
Similarly, the purified poxvirus vector rvv-. DELTA.H 3L/G was seeded onto Vero cells (1X 10)7Cells) were observed after 48 hours, and when 80% or more of the cells were infected, the cells were collected, centrifuged at 1800g for 5 minutes, and the supernatant was removed. SDS-PAGE samples were then prepared and immunoblot hybridization experiments were performed, and incubated with anti-H3L-antibody (D67, see U.S. Pat. No. 9447172) and rabbit anti-human-HRP (Boster, BA1070) as a secondary antibody, as shown in FIG. 4, vaccinia virus wild-type rvv-WT showed significant H3L expression at 35kD, consistent with the expected molecular weight location, while Vero cell control and recombinant vaccinia virus vector rvv-. DELTA.H 3L/G showed no H3L expression, indicating that the H3L gene was knocked out.
Example 4 recombinant vaccinia virus vector rvv- Δ H3L/G amplification preparation and titration.
The recombinant vaccinia virus vector rvv- Δ H3L/G and the vaccinia virus wild strain (rvv-WT) constructed in example 2 were amplified on Vero cells, respectively, as follows:
on the previous day, Vero monolayers (1X 10) with 100% confluency were prepared7Cells/dish) for a total of 10 dishes.
The supernatant was removed and replaced with maintenance medium, the poxvirus to be amplified was inoculated onto the cells (0.01 PFU/cell) and incubated in an incubator at 37 ℃ for 2-3 days, and the lesions were observed.
The cells were scraped and collected, 1800g centrifuged for 5 minutes and the supernatant removed.
Resuspending with 5mL of maintenance medium, and sonicating with a sonicator cell disruptor on ice under the following conditions: 50 watts, 5 seconds sonication/5 seconds interval for 15 minutes.
Repeatedly freezing and thawing twice (about 5 minutes at 80 ℃ in a refrigerator and about 2 minutes at room temperature), and finally oscillating and uniformly mixing; subpackaging in a secondary biological safety cabinet into 1.5mL centrifuge tubes, and freezing at-80 ℃ for 1 mL/tube.
The infectious titer of the amplified vaccinia virus is titrated on Vero cells by the specific method as follows:
vero cells, 3X 10, with a 100% confluency were prepared in 24-well plates on the previous day5A hole.
Remove supernatant and add 200 μ L of maintenance medium per well to prevent cells from drying out.
Adding 100 mu L poxvirus to be detected into 900 mu L maintenance medium, ten-fold diluting, and continuously diluting 10 times1，102，103… …, up to 109And (4) doubling. Note that: when dilution is performed, the tip should be replaced every time dilution is performed from high concentration to low concentration.
From small to large virus concentration (10)9，108，……104) Added to a 24-well plate at 400. mu.L dilution per well, and repeated two times, and the dilution was measured at 6 times in series. The added 24-well plate was incubated in a 37 ℃ cell incubator for 2 days.
The number of viral plaques, more than 20, was counted microscopically and scored as 20 +. Two replicate wells with a measurable number of 20 spots (including 20) were averaged by a dilution of 2.5 (1000. mu.L/400. mu.L). times.corresponding wells to obtain the recombinant virus titer (PFU/mL). The titer of vaccinia virus vectors was titrated as shown in Table 4.
TABLE 4 vaccinia Virus vector titer titration
| Vaccinia virus | Potency (PFU/mL) |
| Vaccinia virus wild type rvv- |
1×108 |
| Recombinant vaccinia virus rvv-delta H3L/ |
1×108 |
Example 5 detection of neutralizing antibodies.
The detection method of the neutralizing antibody comprises the following steps: a96-well flat-bottom plate was prepared by adding 100. mu.L of DMEM cell culture medium to all wells in column 1 (cell control) and 50. mu.L of LDMEM cell culture medium to all wells in columns 2-12 (column 2 will be used as virus control). mu.L of DMEM cell culture medium (1: 10 initial dilution, 2-fold gradient dilution) was added to wells H3-H12. 20 mu L of human plasma samples to be tested are respectively added into H3-H12 wells. Mix all samples in row H and then transfer 50. mu.L to row G. Mixing and transfer were repeated until row a (2-fold gradient dilution). After the final transfer and mixing was complete, 50. mu.L was discarded from row A, columns 3-12. The amount of vaccinia virus was calculated and 40 PFU/well was used as the infectious dose to thaw sufficient viral stocks. When the thawing was complete, the virus was diluted to 800PFU/mL with DMEM cell culture medium. 50 μ L of virus dilution was added to all wells in 2-12 columns in a 96 well plate, the plate was covered, and incubated for 1 hour in a cell incubator. Preparing Vero cells, digesting Vero cells with pancreatin/EDTA to obtain cell suspension, and maintaining culture medium with 2% DMEM to adjust cell concentration to 5 × 105mL, then added to a 96 well cell culture plate at 100. mu.L/well, i.e., 5X 104A hole. After 48 hours, the supernatant was carefully aspirated, 0.1% crystal violet was added, 50 μ L/well, and stained for 10 minutes at room temperature. Photograph, count the number of plaques in each well, divide the number of plaques in the experimental well by the mean of the plaques in the virus control (column 2), and thenThe percent inhibition of the sample was determined by subtracting 1 and then multiplying by 100. IC50 titers were determined as percent inhibition, and neutralizing antibody IC50 titers were expressed as percent inhibition>The reciprocal of the highest dilution of 50, the IC50 can also be calculated from a percentage inhibition fit curve.
As shown in FIG. 5, human plasma showed a high level of neutralizing antibodies against vaccinia virus wild type (rvv-WT) (mean value of GMT 113.1), while the level of neutralizing antibodies against H3L defective strain rvv-. DELTA.H 3L/G was significantly reduced (mean value of GMT 31.8, p < 0.001). The result shows that the strain subjected to H3L knockout generates escape for existing antibodies in human bodies, so that the chance of reinfection is increased, and the application possibility of the vaccinia virus vector is improved.
Example 6 kinetics of viral replication
Vero cells were prepared in 6-well plates and vaccinia virus was inoculated to each well (1.5X 10) at 0.1MOI5PFU), supernatants and cells were then taken at various time points post infection, frozen and thawed 3 times and titrated on Vero cells (see method for virus titration in example 4).
As a result, as shown in FIG. 6, the wild-type vaccinia virus rvv-WT had a strong replication ability, and the virus was mainly present in the cells and contained a small amount of supernatant, which was about 2 orders of magnitude lower than that of the cells. Compared with the wild-type strain, the H3L defective strain rvv-delta H3L/G has weak replication capacity and consistent trend of replication kinetic curves, all reach a peak at 48 hours and are about 2 orders of magnitude lower than the wild-type rvv-WT, and then the difference gradually reduces at a later time point. The H3L knock-out was shown to affect the replication capacity of vaccinia virus.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (16)
1. A recombinant vaccinia virus vector with a H3L gene knocked out, wherein a gene encoding H3L of the recombinant vaccinia virus vector is knocked out, and the H3L gene cannot be normally expressed.
2.The recombinant vaccinia virus vector with H3L knockout function according to claim 1, wherein the vaccinia virus vector is a replicating vaccinia virus vector or a non-replicating vaccinia virus vector.
3. The recombinant vaccinia virus vector with a knockout of H3L gene of claim 2, wherein the H3L knockout is performed by inserting a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene into the nucleic acid sequence at both ends of H3L, so that the H3L gene is mutated, and the nucleic acid sequence further comprises one or more exogenous genes besides the reporter gene or the resistance gene.
4. The recombinant vaccinia virus vector for knocking out H3L gene according to claim 3, wherein the vaccinia virus promoter element is P7.5 or PE/L, preferably the nucleic acid sequence of P7.5 is SEQ ID NO. 13, and the nucleic acid sequence of PE/L is SEQ ID NO. 14.
5. The recombinant vaccinia virus vector with a knockout of H3L gene of claim 4, wherein the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene.
6. The recombinant vaccinia virus vector of knockout of H3L gene of claim 3, wherein the nucleic acid sequences at both ends of H3L are H3L-L homologous recombination arm or H3L-R homologous recombination arm, respectively, wherein the nucleic acid sequence of the H3L-L homologous recombination arm is SEQ ID NO. 1, and the nucleic acid sequence of the H3L-R homologous recombination arm is SEQ ID NO. 2.
7. The recombinant vaccinia virus vector with a knockout of H3L gene of claim 6, wherein the recombinant vaccinia virus vector with a knockout of H3L is rvv- Δ H3L/G.
8. A shuttle vector for constructing a recombinant vaccinia virus vector with an H3L gene knockout, wherein the shuttle vector comprises an H3L homologous recombination arm sequence.
9. The shuttle vector of claim 8, wherein the shuttle vector comprises an H3L-L homologous recombination arm sequence, wherein the H3L-L homologous recombination arm nucleic acid sequence is SEQ ID No. 1.
10. The shuttle vector of claim 9, further comprising an H3L-R homologous recombination arm sequence, wherein the H3L-R homologous recombination arm nucleic acid sequence is SEQ ID No. 2.
11. A shuttle vector according to claim 10 wherein a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, is inserted between the H3L homologous recombination arm sequences, and wherein the nucleic acid sequence further comprises one or more foreign genes other than the reporter gene or the resistance gene.
12. The shuttle vector of claim 11, wherein the vaccinia virus promoter element is P7.5 or PE/L, preferably the nucleic acid sequence of P7.5 is SEQ ID NO. 13, and the nucleic acid sequence of PE/L is SEQ ID NO. 14.
13. The shuttle vector of claim 12, wherein the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene.
14. The shuttle vector of claim 13, wherein the shuttle vector is pΔ H3L-GFP, and the nucleic acid sequence is SEQ ID NO 17.
15. A preparation method for constructing a recombinant vaccinia virus vector with an H3L gene knockout function is characterized by comprising the following steps:
s1, designing primers, and amplifying two homologous recombination arms of H3L by PCR respectively;
s2, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the H3L homologous recombination arm sequence;
s3, transfecting the shuttle vector into a vaccinia virus infected cell;
s4, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on the new cells.
16. The method according to claim 15, wherein the primer is a primer sequence capable of binding to both ends of H3L, and preferably comprises the following nucleic acid sequence:
3H3L-LC-F primer of SEQ ID NO
TATAACCAGACCGTTCAGACTCTTGTCATTATAGTGGGA
Primer SEQ ID NO. 4H3L-LC-R
ATTAATTTTTATCGATCTGCCAAATATGTAGAACACGA
Primer SEQ ID NO 5H3L-RC-F
GTTATGCGGCCGCGGATCTGGCATAACTTCGTTGTCC
6H3L-RC-R primer
TTAATAATCTCATTTCAGAACGGTAGAAGAATCCACTC。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110679461.6A CN113481241A (en) | 2021-06-18 | 2021-06-18 | Recombinant vaccinia virus vector with H3L gene knocked out |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110679461.6A CN113481241A (en) | 2021-06-18 | 2021-06-18 | Recombinant vaccinia virus vector with H3L gene knocked out |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113481241A true CN113481241A (en) | 2021-10-08 |
Family
ID=77935559
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110679461.6A Pending CN113481241A (en) | 2021-06-18 | 2021-06-18 | Recombinant vaccinia virus vector with H3L gene knocked out |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113481241A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654666A (en) * | 2005-01-11 | 2005-08-17 | 武汉大学 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
| CN102206679A (en) * | 2011-03-30 | 2011-10-05 | 中国人民解放军军事医学科学院军事兽医研究所 | Shuttle vector of vaccinia virus and its application |
| CN105861558A (en) * | 2016-06-24 | 2016-08-17 | 西安医学院 | Vaccinia virus shuttle vector and preparation method and application thereof |
| CN109810953A (en) * | 2019-01-07 | 2019-05-28 | 西安彤盛生物科技有限公司 | The recombination the Temple of Heaven strain oncolytic vaccinia virus of removal TK gene and its preparation and application |
| WO2020086423A1 (en) * | 2018-10-22 | 2020-04-30 | Icell Kealex Therapeutics | Mutant vaccinia viruses and use thereof |
-
2021
- 2021-06-18 CN CN202110679461.6A patent/CN113481241A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654666A (en) * | 2005-01-11 | 2005-08-17 | 武汉大学 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
| CN102206679A (en) * | 2011-03-30 | 2011-10-05 | 中国人民解放军军事医学科学院军事兽医研究所 | Shuttle vector of vaccinia virus and its application |
| CN105861558A (en) * | 2016-06-24 | 2016-08-17 | 西安医学院 | Vaccinia virus shuttle vector and preparation method and application thereof |
| WO2020086423A1 (en) * | 2018-10-22 | 2020-04-30 | Icell Kealex Therapeutics | Mutant vaccinia viruses and use thereof |
| CN109810953A (en) * | 2019-01-07 | 2019-05-28 | 西安彤盛生物科技有限公司 | The recombination the Temple of Heaven strain oncolytic vaccinia virus of removal TK gene and its preparation and application |
Non-Patent Citations (1)
| Title |
|---|
| KEVIN SONG,等: "Design and Engineering of Deimmunized Vaccinia Viral Vectors", 《BIOMEDICINES》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Staib et al. | Construction and isolation of recombinant MVA | |
| EP0515433A1 (en) | Recombinant poxvirus internal cores. | |
| US11033617B2 (en) | Duck hepatitis A virus type 3 mutant CH-P60-117C and construction thereof | |
| Staib et al. | Improved host range selection for recombinant modified vaccinia virus Ankara | |
| CN108220251B (en) | Recombinant infectious pustulosis virus, and preparation method and application thereof | |
| CN111100845B (en) | Recombinant measles virus and preparation method and application thereof | |
| Zhu et al. | The attenuation of vaccinia Tian Tan strain by the removal of the viral M1L-K2L genes | |
| CN114015660A (en) | Construction of ten-gene-deleted attenuated African swine fever virus strain and application of ten-gene-deleted attenuated African swine fever virus strain as vaccine | |
| JP2005525119A (en) | Recombinant fowlpox virus | |
| US20240043870A1 (en) | Modified parapoxvirus having increased immunogenicity | |
| CN113481241A (en) | Recombinant vaccinia virus vector with H3L gene knocked out | |
| CN113322279A (en) | Recombinant vaccinia virus vector with D8L gene knocked out | |
| CN113481240A (en) | Recombinant vaccinia virus vector with L1R gene knocked out | |
| CN113322280A (en) | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo | |
| CN114058645A (en) | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo | |
| CN114058643A (en) | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo | |
| CN114058644A (en) | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo | |
| EP4014991A1 (en) | Modified parapoxvirus having increased immunogenicity | |
| CN112638412A (en) | Methods and compositions for producing viruses | |
| CN116515775B (en) | Pseudorabies virus with envelope expressing porcine circovirus 2 capsid protein and application thereof | |
| CN114717204A (en) | Replication-defective pseudorabies virus and construction method and application thereof | |
| WO2013011179A2 (en) | Recombinant vectors based on the modified vaccinia ankara (mva) virus, with deletion in the c6l gene, as vaccines against hiv/aids and other diseases | |
| CN117737008A (en) | Recombinant sheep mouth sore virus expressing sheep pox virus P32 gene and preparation method and application thereof | |
| CN111450247A (en) | Application of L T as duck tembusu virus subunit vaccine adjuvant | |
| CN117327736A (en) | Recombinant vector vaccine for expressing novel coronavirus S protein, recombinant virus vaccine and application |
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: 20211008 |