WO2022218325A1 - 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用 - Google Patents

一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用 Download PDF

Info

Publication number
WO2022218325A1
WO2022218325A1 PCT/CN2022/086526 CN2022086526W WO2022218325A1 WO 2022218325 A1 WO2022218325 A1 WO 2022218325A1 CN 2022086526 W CN2022086526 W CN 2022086526W WO 2022218325 A1 WO2022218325 A1 WO 2022218325A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
swine fever
african swine
fever virus
virus
Prior art date
Application number
PCT/CN2022/086526
Other languages
English (en)
French (fr)
Inventor
宋庆庆
叶正琴
陈鸿军
王衡
狄栋栋
武瑾贤
龚浪
屠颉
谢振华
陈坚
刘建奇
徐丽媛
赵丽霞
刘英楠
张翀宇
Original Assignee
金宇保灵生物药品有限公司
华南农业大学
中国农业科学院上海兽医研究所
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 金宇保灵生物药品有限公司, 华南农业大学, 中国农业科学院上海兽医研究所 filed Critical 金宇保灵生物药品有限公司
Publication of WO2022218325A1 publication Critical patent/WO2022218325A1/zh

Links

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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • C12N2710/12011—Asfarviridae
    • C12N2710/12021—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
    • C12N2710/12011—Asfarviridae
    • C12N2710/12022—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
    • C12N2710/12011—Asfarviridae
    • C12N2710/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/12011—Asfarviridae
    • C12N2710/12051—Methods of production or purification of viral material
    • C12N2710/12052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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
    • C12N2710/12011—Asfarviridae
    • C12N2710/12061—Methods of inactivation or attenuation
    • C12N2710/12062—Methods of inactivation or attenuation by genetic engineering
    • 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

Definitions

  • the invention belongs to the technical field of biological vaccine products, in particular to a gene-deleted attenuated African swine fever virus strain and a construction method and application thereof, in particular to a simultaneous deletion of CD2v, MGF (12L, 13L, 14L) and I177L genes Partially fragmented attenuated African swine fever virus strain and its construction method and application in vaccine preparation.
  • African Swine Fever Virus is the only African virus genus in the African family of viruses, and the only DNA arbovirus genus at present.
  • the virus is mainly infected by direct contact with the mouth or nasal cavity, and has multiple circulation modes of transmission. It mainly exists in blood, tissue fluid, internal organs, secretions and excrement. It has the characteristics of high infection rate, rapid spread and high fatality rate. The pig industry has caused huge economic losses.
  • the main method of prevention against the virus is vaccination.
  • African swine fever virus there are no vaccine products available on the market.
  • inactivated African swine fever virus vaccines live attenuated vaccines and genetically engineered vaccines, none of them have achieved the expected results after effect evaluation. Immune effect.
  • inactivated vaccines cannot produce neutralizing antibodies in animals, so they cannot generate immunity against virulent attacks; live attenuated vaccines have many immune side effects and are prone to multiple infections, which have certain potential biological safety hazards; prepared by genetic engineering The DNA vaccine can obtain a certain degree of immune protection in animals, but the corresponding antibodies were not detected, and it is speculated that cellular immunity may dominate.
  • Patent document CN110551695A (hereinafter referred to as document 1) has disclosed an attenuated vaccine strain with simultaneous deletion of CD2v and MGF genes (MGF360-12L, MGF-13L and MGF-14L), and the weakening of its virulence is mainly caused by the deletion of MGF-360 gene and showed good immune protection.
  • the piglets were inoculated with the gene deletion vaccine strain disclosed in this document 1, although the piglets did not show abnormal adverse reactions, the piglets would have a transient increase in body temperature on the 2nd to 3rd days after the inoculation, and the highest body temperature could reach 41.3°C. There are potential security issues.
  • MGF360-12L, MGF-13L and MGF-14L are near and itself is a fragment that is relatively easy to recombine.
  • Existing studies have also shown that the deletion site caused by the subculture of the wild strain of the virus on the passaged cells, or the deletion of the natural deletion strain directly isolated in nature. The points are basically near these MGF genes, see for example Chapman DA, Tcherepanov V, Upton C, Dixon LK. 2008. Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates.
  • MGF gene fragments such as MGF360-12L, MGF-13L, and MGF-14L, which are prone to self-mutation and homologous recombination deletion or abolishment of deletion sites, are very likely to cause the deletion disclosed in the above-mentioned document 1.
  • one aspect of the present invention provides a gene-deleted attenuated African swine fever virus strain, which has the following genes or fragments deleted in the genome of type II African swine fever virus:
  • CD2V gene MGF360 (12L, 13L, 14L) and I177L gene fragment, wherein said MGF360 (12L, 13L, 14L) represents MGF360-12L gene, MGF360-13L gene and MGF360-14L gene, the nucleus of said I177L gene fragment
  • the nucleotide sequence is shown in SEQ ID NO:14.
  • the above-mentioned type II African swine fever virus includes but is not limited to ASFV-SY18, Georgia 2008/1, Pig/HLJ/2018, Georgia 2007/1, ASFV GZ2018.
  • Another aspect of the present invention also provides a method for constructing an attenuated African swine fever virus strain, comprising the steps of: genetically engineering the CD2V gene of parent type II African swine fever virus, MGF360 (12L, 13L, 14L) The gene and the I177L gene fragment are deleted, wherein the nucleotide sequence of the I177L gene fragment is shown in SEQ ID NO: 14.
  • the genetic engineering means is homologous recombination technology, which specifically includes the following steps:
  • step S4) Transfect the first homologous recombination transfer vector of step S1) into the primary PAM cells infected with the parental type II African swine fever virus with a transfection reagent, and obtain deletion through the first screening expression cassette selection
  • the second homologous recombination transfer vector of step S2) is transfected into the primary PAM cells infected with the first recombinant virus obtained in step S4) with a transfection reagent, and the expression cassette and the second Screening the expression cassette to obtain the second recombinant virus with deletion of MGF360 (12L, 13L, 14L) gene and CD2V gene at the same time;
  • step S6 transfecting the third homologous recombination transfer vector in step S3) with a transfection reagent into the primary PAM cells infected with the second recombinant virus obtained in step S5), screening the expression cassette, the second The screened expression cassette and the third screened expression cassette were screened to obtain a third recombinant virus that simultaneously deleted the MGF360 (12L, 13L, 14L) gene, CD2V gene and I177L gene fragment as an attenuated African swine fever virus strain.
  • the first screening expression cassette, the second screening expression cassette and the third screening expression cassette are different screening expression cassettes, all of which are selected from mCherry, EGFP and EBFP expression cassettes.
  • Another aspect of the present invention also provides a live attenuated African swine fever virus vaccine, which is prepared from the above-mentioned gene-deleted attenuated African swine fever virus strain.
  • the virus content of the attenuated African swine fever virus is greater than or equal to 10 7.0 TCID 50 /ml.
  • Another aspect of the present invention also provides the preparation method of the above-mentioned African swine fever virus attenuated live vaccine, which comprises the following steps:
  • T1 inoculate primary PAM cells with the attenuated African swine fever virus strain of the above-mentioned gene deletion, expand the culture, and harvest the virus liquid;
  • a vaccine is prepared directly or with an adjuvant from a virus liquid with a virus content of ⁇ 10 7.0 TCID 50 /ml (for example, it can be obtained by concentrating the virus liquid harvested in step T1).
  • the adjuvant includes but is not limited to nano-adjuvant, interleukin and interferon.
  • the gene-deleted attenuated African swine fever virus strain provided by the above technical solution is based on the deletion of the MGF360 (12L, 13L, 14L) gene and the CD2v gene, and also lacks a partial fragment of the I177L gene, and obtains a relative to the wild virus strain.
  • the strain is significantly attenuated (the double attenuation effect of MGF360 (12L, 13L, 14L) gene deletion and I177L gene partial fragment deletion), and can efficiently and stably propagate, maintain high virulence and immunogenicity, and can be distinguished from wild A strain of African swine fever virus (rASFV GZ2018 ⁇ MGF360/CD2v/I177L) that exploits the deletion of the CD2v gene.
  • the present invention was the simultaneous deletion of two virulence genes (I177L gene fragment and MGF360 (12L, 13L, 14L) gene) , in which the I177L gene has only a single gene in the entire genome, which is not as easy to be replicated or recombined multiple times as multi-gene family genes (such as the MGF360 gene), and the phenomenon of deletion complementation occurs because the I177L gene fragment is deleted alone.
  • Figure 1 is a fluorescent photo of blue protein expression after the deletion of the African swine fever virus I177L gene fragment, in which panel A represents the fluorescent photo after the deletion of the African swine fever virus I177L-D gene fragment, and panel B represents the African swine fever virus I177L-A and The fluorescent photo after the deletion of the I177L-B gene fragment, the D panel represents the fluorescent photo after the deletion of the I177L-C gene fragment of African swine fever virus;
  • Fig. 2 is the electrophoresis picture of homologous recombination transfer vector PCR identification
  • Figure 3 is a fluorescent photo of red protein expression after the deletion of the African swine fever virus MGF360 (12L, 13L, 14L) gene;
  • Fig. 4 is the electrophoresis picture of recombinant virus PCR identification
  • Figure 5 is a fluorescent photo of green protein expression after the deletion of the CD2V gene of African swine fever virus.
  • the present invention aims to provide an African swine fever with higher safety
  • the virus attenuated gene deletion vaccine strain has good safety and no potential risk of virulence returning to strong.
  • the invention also provides a construction method of the gene deletion vaccine strain and a live attenuated African swine fever virus vaccine based on the gene deletion vaccine strain with better safety and immune protection ability.
  • Example 1 Isolation and identification of African swine fever virus strains
  • the African swine fever virus wild strain used in the embodiment is the Guangdong strain isolated in the wild by the Department of Avian Diseases, School of Veterinary Medicine, South China Agricultural University, named ASFV GZ2018, and the specific operation includes the following steps (operated in a biosafety level 3 laboratory. ): Weigh 0.1 g of the spleen tissue of the dead pigs detected as positive ASFV infection, put it into a 2.0 mL sterile EP tube, add 1 mL of sterile PBS solution to the tube, and then put 3 sterile tissue grinders Steel balls, put the EP tube into the tissue grinder, and perform oscillating grinding. Note that the grinder is pre-cooled to 4 °C in advance and placed in a balanced position.
  • CPE cytopathic effect
  • the identification of the ASFV GZ2018 strain was completed by the Department of Poultry Diseases, School of Veterinary Medicine, South China Agricultural University. Specifically, the genome data of the strain was obtained through genome sequencing (Genebank accession number is MT496893), and its genome sequence was compared with the existing ASFV in Genebank. Genomic data (e.g.
  • ASFV type II strain ASFV-SY18 whose Genebank accession number is MH713612; ASFV type II strain Georgia 2008/1, whose Genebank accession number is MH910495; ASFV type II strain Pig/HLJ/2018, whose Genebank accession number is MH910495;
  • the accession number is MK333180; the type I ASFV strain Ba71V, whose Genebank accession number is FJ174348, etc.) are compared, and it is proved that the ASFV GZ2018 strain is a genotype II ASFV wild strain.
  • the ASFV GZ2018 strain was approved and transported to the biosafety level 3 laboratory of Jinyu Baoling Biopharmaceutical Co., Ltd.
  • this document 2 does not specifically disclose the position of the deleted I177L gene fragment; and because the gene is very short, the expressed protein will not stimulate the organism to produce corresponding antibodies, so the African swine fever virus gene deletion strain constructed by the above document 2 After inoculating piglets, it is impossible to distinguish whether it is the antibody produced by the vaccination or the antibody produced by the wild virus infection, which is very unfavorable for the prevention, control and purification of African swine fever virus.
  • the I177L gene (its CDS region is 174513-174713 in MT496893 (201 bp in total), and its ORF region is 174513-175043 in MT496893 (531 bp in total)) is also the start region of its downstream genes, so the complete Deletion or deletion of certain segments may result in the inability of downstream genes to be expressed, which may lead to the inability of gene-deleted strains of the virus to replicate stably or to lose the immunogenicity of the virus.
  • the inventor first constructed a variety of I177L gene fragments of the strain (including Nos. 174513-174713 in MT496893 (201bp in total, named I177L-A) on the basis of the GZ2018 strain obtained in Example 1 above.
  • the nucleotide sequence is shown in SEQ ID NO: 11), the 174513-175043 (531bp in total, named I177L-B, its nucleotide sequence is shown in SEQ ID NO: 12), the 174516-174599 (a total of 84bp, Named I177L-C, whose nucleotide sequence is shown in SEQ ID NO: 13), the 174600-174710 (total 111bp, named I177L-D, whose nucleotide sequence is shown in SEQ ID NO: 14) deletion
  • the gene-deleted strains were selected to determine the appropriate I177L gene deletion fragments, without affecting the reproductive stability of the constructed gene-deletion strains. The specific operations included the following steps.
  • step 2.1.2 Using the same method as step 2.1.1, the homologous recombination transfer vectors with deletions of I177L-B, I177L-C and I177L-D were constructed respectively, named pBlue-LR- ⁇ I177L-B-EBFP, pBlue- LR- ⁇ I177L-C-EBFP and pBlue-LR- ⁇ I177L-D-EBFP.
  • Primary PAM cells were infected with ASFV GZ2018 wild strain, and the homologous recombination transfer vectors pBlue-LR- ⁇ I177L-A-EBFP, pBlue-LR- ⁇ I177L-B-EBFP, pBlue-LR- ⁇ I177L-C- EBFP and pBlue-LR- ⁇ I177L-D-EBFP were transfected with TurboFectin 8.0 transfection reagent (purchased from Origene, USA), respectively, to transfect primary PAM cells infected with wild strains, and cultured at 37°C for 48h (the medium was PRMI1640 medium containing 10% FBS), and observe whether there is blue fluorescence signal under the microscope.
  • TurboFectin 8.0 transfection reagent purchased from Origene, USA
  • the present invention determines the deletion of the I177L-D gene fragment, namely the 174600-174710 nucleotides (111bp in total) in the MT496893 of the ASFV GZ2018 strain, and the deletion of this fragment will not affect the replication ability of the constructed gene deletion recombinant virus,
  • the gene deletion recombinant virus obtained by construction was named rASFV GZ2018 ⁇ I177L-D.
  • Example 3 Construction of recombinant virus deletion of MGF360 (12L, 13L, 14L), CD2V and I177L-D genes
  • the inventors attempted to construct a recombinant virus that simultaneously deleted MGF360 (12L, 13L, 14L), CD2V and I177L-D genes from the ASFV GZ2018 strain, and verified whether a stably replicated gene-deleted recombinant virus could be obtained, specifically Include the following steps.
  • the source arm was directional cloned into pBluescript II KS vector to obtain a homologous recombination transfer vector lacking MGF360 (12L, 13L, 14L) gene, named pBlue-LR- ⁇ MGF360-P72- mCherry (full length 3301bp), lanes 1-3 in Figure 2 show the identification gel electrophoresis image of the recombinant transfer vector (wherein M represents DL5000 Marker, - represents negative control, + represents empty vector pBluescript II KS control);
  • CD2V-deficient homologous recombination transfer vector Using the method of homologous recombination, the left and right homology arms of the part of the gene to be deleted, namely the left homology arm of CD2V and the right homology arm of CD2V, are combined with the EGFP gene sequence.
  • the homology arm was synthesized, named CD2V-EGFP, and it was cloned into pBluescript II KS vector to obtain homologous recombination deletion of CD2V gene
  • the transfer vector named pBlue-LR- ⁇ CD2V-EGFP (full length 2871bp), in Figure 2, lanes 4-6 show the identification gel electrophoresis of the recombinant transfer vector;
  • rASFV GZ2018 ⁇ MGF360 which lacks the complete reading frame of MGF360 (12L, 13L, 14L) and the MGF360 (12L, 13L, 14L) gene relative to the ASFV GZ2018 wild strain PCR identification of the recombinant virus (identification of MGF360 (12L, 13L, 14L) gene deletion using primers: 5'-aacatgcggtacacacgatg-3' (SEQ ID NO: 5) and 5'-acggccagcaacaaagttt-3' (SEQ ID NO: 6)) The results are shown in lanes 1-3 in Figure 4, where M represents DL5000 Marker, lane 1 represents positive control (ASFV GZ2018 wild strain), lane 2 represents recombinant virus rASFV GZ2018 ⁇ MGF360, and lane 3 represents negative control.
  • step 3.2.4 Infect primary PAM cells with rASFV GZ2018 ⁇ CD2V obtained in step 3.2.2, and transfect PAM cells with pBlue-LR- ⁇ I177L-D-EBFP constructed in step 3.1.3 according to the same method as in step 3.2.1 above, The blue and green double fluorescent spots were repeatedly picked under the microscope for purification, and finally the recombinant African swine fever virus expressing blue and green double fluorescent protein I177L-D and CD2V genes were deleted, named rASFV GZ2018 ⁇ CD2V/ ⁇ I177L-D, It has the CD2V and I177L-D reading frames deleted relative to the ASFV GZ2018 wild strain.
  • step 3.2.5 Infect primary PAM cells with rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V obtained in step 3.2.3, and transfect PAM with pBlue-LR- ⁇ I177L-D-EBFP constructed in step 3.1.3 according to the same method as in step 3.2.1 above cells, and repeatedly picked red, green, and blue fluorescent spots under the microscope for purification, and finally obtained MGF360 (12L, 13L, 14L), CD2V and I177L-D genes that express red, green, and blue fluorescent proteins.
  • a recombinant African swine fever virus named rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D, which has deleted the reading frames of MGF360 (12L, 13L, 14L), CD2V and I177L-D relative to the wild ASFV GZ2018 strain, identified by PCR (identification of I177L
  • the primers used for -D gene deletion are: 5'-gtgggccccttaagatcaca-3' (SEQ ID NO: 9) and 5'-ccactctgatactccccagc-3' (SEQ ID NO: 10))
  • the results are shown in lanes 7-9 in Figure 4 , where lane 7 represents the recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D, lane 8 represents the positive control (ie rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V recombinant virus), and lane 9 represents the negative control
  • the sequencing results showed that the recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D which simultaneously deleted MGF360 (12L, 13L, 14L), CD2V and I177L-D was successfully constructed in Example 3, and the recombinant virus could be expressed in primary PAM cells. Stable replication and reproduction, it can express red, green, and blue fluorescence when infecting PAM cells, and there is no problem that the recombinant virus cannot replicate and reproduce due to the deletion of various virulence genes.
  • Example 4 Titer determination of recombinant virus
  • the titers of the recombinant virus rASFV GZ2018 ⁇ I177L-D constructed in the above Example 2 the recombinant virus rASFV GZ2018 ⁇ CD2V/ ⁇ I177L-D constructed in the above Example 3, and the recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D were measured, and compared with The titers of the wild strain ASFV GZ2018 were compared.
  • the titer of African swine fever virus was determined by two methods: TCID 50 of the half-cell infection dose and half of the hemoglobin 50 of HAD 50 , in which:
  • the TCID 50 titration was carried out according to the following steps: ASFV was serially diluted 10 times in serum-free 1640 medium, inoculated in 96-well culture plates, and PAM cells with a density of about 90-100% were inoculated into 8 wells for each dilution. 100 ⁇ l/well, cultured at 37°C and 5% CO 2 , observed for 3-7 days, and calculated the half-number cell infection dose (TCID 50 ) according to cytopathic or fluorescence changes and Reed and Muench method.
  • the HAD 50 test operation was performed in accordance with the "African Swine Fever Virus Red Blood Cell Adsorption Test Operating Procedures" with appropriate adjustments: inoculate primary PAM cells in a 96-well cell culture plate, and dilute the sample to be tested by 10-fold gradient, 20 ⁇ l/well , virus infection can be judged according to the rosettes formed by red blood cells aggregated around infected cells, observed for 7 days, and calculated the half-hematosorbed dose (HAD 50 ) according to the Reed and Muench method.
  • the specific measurement operation includes the following steps.
  • the porcine primary PAM cells were plated into a 6-well plate, and after growing into a monolayer, 0.1 MOI virus solution (ASFV GZ2018, rASFV GZ2018 ⁇ I177L-D, rASFV GZ2018 ⁇ CD2V/ ⁇ I177L-D or rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D) was inoculated respectively.
  • D incubate at 37°C for 2h, harvest the amplified virus at 0h, 6h, 12h, 18h, 24h, 30h, 36h, 48h, and 72h after infection, repeat freeze-thaw 3 times, centrifuge at high speed, take the supernatant, and obtain the culture respectively.
  • virus fluid (ASFV GZ2018, rASFV GZ2018 ⁇ I177L-D, rASFV GZ2018 ⁇ CD2V/ ⁇ I177L-D or rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/
  • the viral titers of D and rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D were 10 7.20 TCID 50 /mL, 10 4.30 TCID 50 /mL, 10 4.28 TCID 50 /mL, and 10 4.25 TCID 50 /mL, respectively;
  • the virus titers of ASFV GZ2018, rASFV GZ2018 ⁇ I177L-D, rASFV GZ2018 ⁇ CD2V/ ⁇ I177L-D and rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D were 10 7.50 TCID 50 /mL, 10 5.80 TCID 50 /mL, 10 5.65 TCID 5 /mL, respectively 10 5.25 TCID50 /mL.
  • the titer of the gene deletion recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D is significantly lower than that of the wild strain ASFV GZ2018 (the former is 100 to 1000 times lower than the latter), and it is also significantly lower
  • the titer of the SY18 ⁇ MC group virus (deletion of MGF360 (12L, 13L, 14L) and CD2V) disclosed in Document 1 (the average titer at 48 h after infection was 8.472 ⁇ 10 6 TCID 50 /mL), and was similar to that disclosed in Document 2 only.
  • the virulence of the gene-deleted strain ASFV-G- ⁇ I177L, which lacks the I177L gene segment, is comparable, and does not result in low viral titers and immunogenicity of the gene-deleted strain rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D due to the deletion of more virulence genes.
  • the gene deletion recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D constructed and obtained in the present invention is an African swine fever virus gene deletion recombinant virus that is significantly attenuated relative to the wild strain, and still maintains a relatively high virus titer and higher immunogenicity. Theoretically, without reducing immunogenicity, the more virulence genes are deleted, the weaker the virulence of the virus and the lower the safety risk.
  • the present invention provides simultaneous deletion of MGF360 (12L, 13L, 14L), CD2V
  • the recombinant virus with the I177L-D gene will have higher safety than the wild strain, the gene deletion strain disclosed in Document 1 and Document 2, and since I177L is a single gene in the genome, it will not easily undergo self-mutation and homology.
  • the phenomenon of source recombination deletion or complementation of the deletion site can also avoid the potential safety risk of virulence reversion.
  • Example 5 Virulence detection of gene deletion strain rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D
  • the virulence of the gene deletion recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D obtained in the above Example 3 was detected to evaluate its safety, which specifically included the following steps.
  • the piglets in each group were measured for feed intake, body temperature, and body weight changes every day, and the survival of the piglets was recorded until 28 days after inoculation; during the observation period, the peripheral blood of the piglets in each group was collected every 5 days. , saliva, feces and lacrimal glands (if the piglet died, the collection of the dead piglet was stopped) to detect the virus content (HAD 50 ) in the piglet's blood and the presence of detoxification in saliva, feces and lacrimal glands.
  • HAD 50 virus content
  • Table 1 Test protocol for virulence detection of gene deletion strain rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D
  • Example 6 Evaluation of the immune protection effect of gene deletion recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D as a vaccine strain on piglets
  • the gene deletion recombinant virus rASFV GZ2018 ⁇ MGF360/ ⁇ CD2V/ ⁇ I177L-D is used as a vaccine strain to immunize piglets, so as to evaluate its immune protection effect through antibody detection and challenge test, which specifically includes the following steps.
  • Antibody test results showed that: piglets in group a and group b did not produce specific antibodies on days 3, 5, and 7 after immunization; the positive rates of antibodies in group z were 40% on days 14, 21, and 28 after immunization, respectively. , 80% and 100%; the antibody positive rate of piglets in group b was 100% on the 14th, 21st and 28th days after immunization. Piglets in groups c and d did not produce specific antibodies during the observation period. During the observation period, the body temperature of piglets in group a and group b was within the normal temperature range, no abnormal clinical manifestations were found, no viremia occurred, and there was no detoxification in feces, saliva, lacrimal glands and other parts. Necropsy showed that no obvious pathological changes were found in all immunized piglets.
  • the antibody positive rate before the second vaccination was 40% on the 14th day after the first vaccination, and the antibody positive rate continued to increase on the 7th and 14th days after the second vaccination, and the antibody positive rate before the challenge reached 100%.
  • 10 5.0 TCID 50 /head high-dose group can reach 100% antibody positive rate 14 days after the first vaccination and before the second vaccination, and all piglets have body temperature within the normal range, no abnormal clinical manifestations, and no viremia
  • the wild strain ASFV GZ2018 still retains high immunogenicity after deletion of MGF360 (12L, 13L, 14L), CD2v and I177L-D, and can provide effective challenge protection after immunizing healthy susceptible pigs. It can provide safe and effective immune protection, so it can be used as a candidate African swine fever virus vaccine strain.
  • the gene-deleted attenuated African swine fever virus strain constructed by the present invention can be inoculated into primary PAM cells, expanded for culture, and the virus liquid is harvested and the virus titer is determined.
  • virus liquid with a virus content ⁇ 10 7.0 TCID 50 /ml to make a vaccine directly or with adjuvants (such as nano-adjuvant, interleukin or interferon), and the virus content in the prepared vaccine can be as high as 10 7.0 TCID 50 /ml above.
  • adjuvants such as nano-adjuvant, interleukin or interferon
  • the present invention provides a gene-deleted attenuated African swine fever virus strain and a construction method and application thereof.
  • the gene-deleted attenuated African swine fever virus strain is constructed by a homologous recombination method, and is significantly reduced compared to the parent strain. It can be used as a candidate vaccine strain with good safety and immune protection effect, and is suitable for industrial application.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用，属于生物疫苗制品技术领域。本发明通过同源重组方法构建的基因缺失的减毒非洲猪瘟病毒毒株是在II型非洲猪瘟病毒基因组的基础上同时缺失了CD2v，MGF(12L,13L,14L)和I177L基因片段的基因缺失毒株，其相对于亲本毒株明显减毒，且不会影响基因缺失病毒株的稳定复制和免疫原性。将其接种实验猪后不会出现实验猪体温明显升高、关节肿胀、发病或死亡现象，也无病毒血症发生，表现出良好的安全性和良好的免疫攻毒保护效果。因此本发明提供的基因缺失的减毒非洲猪瘟病毒毒株可以作为一种具有良好安全性和免疫保护效果的候选疫苗株。

Description

一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用 技术领域
本发明属于生物疫苗制品技术领域，具体涉及一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用，尤其涉及一种同时缺失CD2v，MGF(12L,13L,14L)和I177L基因部分片段的减毒非洲猪瘟病毒毒株及其构建方法和在疫苗制备中的应用。
背景技术
非洲猪瘟病毒(African Swine Fever Virus，ASFV)是非洲病毒科中唯一的非洲病毒属，也是目前唯一的DNA虫媒病毒属。该病毒主要靠口或鼻腔直接接触感染，同时拥有多个循环传播方式，主要存在于血液、组织液、内脏、分泌物和排泄物中，具有感染率高，传播迅速，致死率高等特点，对全球养猪业造成了巨大经济损失。
针对病毒预防的最主要方法是疫苗接种。然而对于非洲猪瘟病毒，市场上还没有可用的疫苗产品，虽然已经尝试开发了非洲猪瘟病毒灭活疫苗、减毒活疫苗及基因工程疫苗等，但经过效果评价后，均未获得预期的免疫效果。其中灭活疫苗无法使动物体内产生中和抗体，因此不能产生抵抗强毒攻击的免疫力；减毒活疫苗免疫副作用多且易引起多重感染等问题，存在一定的生物安全隐患；通过基因工程制备的DNA疫苗可使动物获得一定的免疫保护，但并未检测到相应抗体，推测可能为细胞免疫占主导地位。
研究表明，在非洲猪瘟病毒编码的蛋白成分中，很多具有免疫抑制特性，例如已经明确的与免疫抑制有关的基因主要为CD2V、MGF和9GL等。因此，目前针对非洲猪瘟病毒疫苗的研究主要集中在利用这些免疫抑制基因缺失的毒株作为疫苗株制备疫苗。其中研究较多的为CD2v和MGF基因缺失株，然而CD2v缺失对毒株的毒力影响有限(几乎没有影响)，MGF基因缺失后不同剂量的研究会引起接种猪体温变化，并且会与非洲猪瘟流行毒株发生重组而返回毒性。专利文献CN110551695A(以下称文献1)已公开一种CD2v和MGF基因(MGF360-12L、MGF-13L和MGF-14L)同时缺失的减毒疫苗株，其毒力减弱主要是MGF-360基因缺失导致的，并且可表现出良好的免疫保护作用。然而，利用该文献1公开的基因缺失疫苗株接种仔猪后，仔猪虽然未显示异常不良反应，但会导致仔猪在接种后第2～3天出现短暂的体温升高，最高体温能达到41.3℃，有潜在的安全性问题。另一方面由于MGF基因属于多基因家族，其中的重复序列很容易通过自身突变和同源重组缺失或回补缺失部位而导致毒力返强，并且MGF360-12L、MGF-13L和MGF-14L附近及本身就是一个比较容易重组的片段，现有研究也表明：该 病毒的野生毒株在传代细胞上进行传代培养导致的缺失位点、或在自然界中直接分离到的自然缺失株所缺失的位点基本上都在这几个MGF基因附近，例如参见Chapman DA,Tcherepanov V,Upton C,Dixon LK.2008.Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates.J Gen Virol 89:397–408；de la Vega I,Vinuela E,Blasco R.1990.Genetic variation and multigene families in African swine fever virus.Virology 179:234-246；Krug PW,Holinka LG,O’Donnell V,Reese B,Sanford B,FernandezSainz I,Gladue DP,Arzt J,Rodriguez L,Risatti GR,Borca MV.2015.The progressive adaptation of a Georgian isolate of African swine fever virus to Vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.J Virol 89:2324-2332.)。因此，MGF360-12L、MGF-13L和MGF-14L等MGF基因片段的这种极易发生自身突变和同源重组缺失或回补缺失部位的现象，极有可能会造成由上述文献1公开的缺失CD2v和MGF360(12L,13L,14L)基因的疫苗株制备的减毒活疫苗面临潜在的毒力返强安全风险。鉴于以上文献1存在的多方面的安全性问题，迫切需要一种安全性更高的非洲猪瘟病毒疫苗产品。
发明内容
针对现有技术中存在的一个或多个问题，本发明一个方面提供一种基因缺失的减毒非洲猪瘟病毒毒株，其在II型非洲猪瘟病毒的基因组中缺失以下基因或片段：
CD2V基因、MGF360(12L,13L,14L)和I177L基因片段，其中所述MGF360(12L,13L,14L)表示MGF360-12L基因、MGF360-13L基因和MGF360-14L基因，所述I177L基因片段的核苷酸序列如SEQ ID NO:14所示。
上述II型非洲猪瘟病毒包括但不限于ASFV-SY18、Georgia 2008/1、Pig/HLJ/2018、Georgia 2007/1、ASFV GZ2018。
本发明另一方面还提供一种减毒非洲猪瘟病毒毒株的构建方法，其包括以下步骤：通过基因工程手段将亲本II型非洲猪瘟病毒的CD2V基因、MGF360(12L,13L,14L)基因和I177L基因片段缺失，其中所述I177L基因片段的核苷酸序列如SEQ ID NO:14所示。
上述方法中，所述基因工程手段为同源重组技术，具体包括以下步骤：
S1)将MGF360(12L,13L,14L)基因的左右同源臂和第一筛选表达盒克隆至pBluescript II KS载体，获得第一同源重组转移载体；
S2)将CD2V基因的左右同源臂和第二筛选表达盒克隆至pBluescript II KS载体，获得第二同源重组转移载体；
S3)将I177L基因片段的左右同源臂和第三筛选表达盒克隆至pBluescript II KS载 体，获得第三同源重组转移载体；
S4)将步骤S1)的第一同源重组转移载体用转染试剂转染至使用所述亲本II型非洲猪瘟病毒感染的原代PAM细胞中，通过所述第一筛选表达盒筛选获得缺失MGF360(12L,13L,14L)基因的第一重组病毒；
S5)将步骤S2)的第二同源重组转移载体用转染试剂转染至使用步骤S4)获得的第一重组病毒感染的原代PAM细胞中，通过所述第一筛选表达盒和第二筛选表达盒筛选获得同时缺失MGF360(12L,13L,14L)基因和CD2V基因的第二重组病毒；
S6)将步骤S3)的第三同源重组转移载体用转染试剂转染至使用步骤S5)获得的第二重组病毒感染的原代PAM细胞中，通过所述第一筛选表达盒、第二筛选表达盒和第三筛选表达盒筛选获得同时缺失MGF360(12L,13L,14L)基因、CD2V基因和I177L基因片段的第三重组病毒，作为减毒非洲猪瘟病毒毒株。
上述方法中，所述第一筛选表达盒、第二筛选表达盒和第三筛选表达盒为不同的筛选表达盒，均选自mCherry、EGFP和EBFP表达盒。
本发明再一方面还提供一种非洲猪瘟病毒减毒活疫苗，其由上述的基因缺失的减毒非洲猪瘟病毒毒株制备获得。
上述非洲猪瘟病毒减毒活疫苗中，减毒非洲猪瘟病毒的病毒含量≥10 7.0TCID 50/ml。
本发明再一方面还提供了上述的非洲猪瘟病毒减毒活疫苗的制备方法，其包括以下步骤：
T1)将上述的基因缺失的减毒非洲猪瘟病毒毒株接种原代PAM细胞，扩大培养，收获病毒液；
T2)以病毒含量≥10 7.0TCID 50/ml的病毒液(例如可以通过浓缩步骤T1收获的病毒液获得)直接或配合佐剂制成疫苗。
上述制备方法中，所述佐剂包括但不限于纳米佐剂、白介素和干扰素。
基于以上技术方案提供的基因缺失的减毒非洲猪瘟病毒毒株在MGF360(12L,13L,14L)基因和CD2v基因缺失的基础上，还缺失了I177L基因部分片段，获得一种相对于野生毒株显著减毒(MGF360(12L,13L,14L)基因缺失和I177L基因部分片段缺失的双重减毒效果)、且能够高效稳定增殖、保持较高的毒力和免疫原性、并可以区别于野生毒株(利用CD2v基因的缺失)的非洲猪瘟病毒毒株(rASFV GZ2018ΔMGF360/CD2v/I177L)。经试验证明，利用本发明提供的rASFV GZ2018ΔMGF360/CD2v/I177L毒株对实验猪进行接种后，按照低剂量(10 3.0TCID 50/头)接种和高剂量(10 5.0TCID 50/头)接种实验猪，均不会出现实验猪体温明显升高(不超 过40.3℃)、关节肿胀、发病或死亡现象，也无病毒血症发生，表现出比文献1和文献2公开的基因缺失株更好的安全性，同时还表现出良好的免疫攻毒保护效果，保护率为100％；另一方面，本发明是两种毒力基因(I177L基因片段和MGF360(12L,13L,14L)基因)的同时缺失，其中I177L基因在整个基因组中仅有单个基因，其不像多基因家族基因(例如MGF360基因)一样容易被多次复制或重组而发生缺失回补的现象，并且由于I177L基因片段单独缺失时就可以表现出显著的减毒效果，即使MGF360(12L,13L,14L)基因发生缺失回补的现象，也不会导致本发明的基因缺失株毒力返强，因此本发明提供的基因缺失株消除了毒力返强的潜在风险。
附图说明
图1为非洲猪瘟病毒I177L基因片段缺失后的蓝色蛋白表达荧光照片，其中A幅表示非洲猪瘟病毒I177L-D基因片段缺失后的荧光照片，B幅表示非洲猪瘟病毒I177L-A和I177L-B基因片段缺失后的荧光照片，D幅表示非洲猪瘟病毒I177L-C基因片段缺失后的荧光照片；
图2为同源重组转移载体PCR鉴定电泳图；
图3为非洲猪瘟病毒MGF360(12L,13L,14L)基因缺失后的红色蛋白表达荧光照片；
图4为重组病毒PCR鉴定电泳图；
图5为非洲猪瘟病毒CD2V基因缺失后的绿色蛋白表达荧光照片。
具体实施方式
针对现有技术中存在的利用基因缺失疫苗株制备的非洲猪瘟病毒减毒活疫苗存在的潜在安全风险和毒力返强安全风险，本发明旨在提供一种安全性更高的非洲猪瘟病毒减毒基因缺失疫苗株，该疫苗株安全性好，且不存在毒力返强的潜在风险。本发明还提供了该基因缺失疫苗株的构建方法和基于该基因缺失疫苗株的安全性和免疫保护能力均较佳的非洲猪瘟病毒减毒活疫苗。
通过以下具体实施方式详细说明本发明。
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《分子克隆实验指南》(《MolecμLar Cloning:A Laboratory Manual》Sambrook，J.，Russell，David W.，MolecμLar Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施 例中的提示替换使用。
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，实施例将有助于理解本发明，但不应作为对本发明内容的限制。
实施例1：非洲猪瘟病毒毒株分离及其鉴定
1.1、毒株
实施例中使用的非洲猪瘟病毒野生毒株为由由华南农业大学兽医学院禽病教研室野外分离的广东毒株，命名为ASFV GZ2018，具体操作包括以下步骤(在生物安全3级实验室中操作)：将检测为阳性ASFV感染的病死猪的脾脏组织称取0.1g，放入2.0mL的无菌EP管中，在管中加入1mL无菌PBS溶液，再放入3粒无菌的组织研磨钢珠，将EP管放入组织研磨仪里，进行震荡研磨，注意将研磨仪提前预冷至4℃并配平放置，将研磨后的组织液反复冻融3次后，用0.45μm的滤膜进行过滤后，接种到培养的原代PAM细胞(华南农业大学兽医学院禽病教研室制备，以10％FBS的PRMI1640培养基(购自Gibco)进行培养)上，能够出现细胞病变效应(CPE)，再进行病毒的分离。
1.2、毒株的鉴定
ASFV GZ2018毒株的鉴定由华南农业大学兽医学院禽病教研室完成，具体为通过基因组测序，获得该毒株的基因组数据(Genebank登录号为MT496893)，并将其基因组序列与Genebank中已有的ASFV基因组数据(例如II型ASFV毒株ASFV-SY18，其Genebank登录号为MH713612；II型ASFV毒株Georgia 2008/1，其Genebank登录号为MH910495；II型ASFV毒株Pig/HLJ/2018，其Genebank登录号为MK333180；I型ASFV毒株Ba71V，其Genebank登录号为FJ174348等)进行比对，证明ASFV GZ2018毒株为基因II型ASFV野毒株。
该ASFV GZ2018毒株通过高致病性病原微生物转运手续审批后运送至金宇保灵生物药品有限公司生物安全3级实验室保存。
实施例2：I177L基因缺失片段的确定
虽然Manuel V.Borca等人(Development of a highly effective African swine fever virus vaccine by deletion of the I177L gene results in sterile immunity against the current epidemic Eurasia strain，Journal of Virology，2020；DOI：10.1128/JVI.02017-19，以下称文献2)已经证明II型非洲猪瘟病毒Georgia 2007/1的基因组中缺失I177L基因片段(Georgia 2007/1的Genebank登录号为NC_044959.1，其中I177L基因片段缺失位置是第174530-174671(共142bp)中的112bp；相当于ASFV GZ2018毒株的基因组MT496893中第174572-174713中的112bp)可以获得安全性和免疫保护效果均良好的基因缺失疫苗 株ASFV-G-ΔI177L。然而，该文献2并没有具体公开缺失的I177L基因片段的位置；并且由于该基因非常短，表达的蛋白不会刺激生物体产生相应的抗体，因此上述文献2构建的非洲猪瘟病毒基因缺失株用于接种仔猪后，无法区分是疫苗接种产生的抗体还是野毒感染产生的抗体，非常不利于非洲猪瘟病毒的防控和净化。虽然可以想到通过进一步缺失其他基因以构建能够区分疫苗株和野毒的多基因(例如I177L和CD2v)缺失疫苗株，但是多种基因的同时缺失还可能会导致病毒毒力过度致弱(尤其是多种毒力基因的同时缺失)，不能实现免疫保护效果，甚至会导致基因缺失株不能稳定复制。另一方面，I177L基因(其CDS区为MT496893中第174513-174713(共计201bp)，ORF区为MT496893中第174513-175043(共计531bp))也是其下游基因的起始区，因此该基因的完全缺失或者某些片段的缺失可能会导致下游基因无法表达，进而可能会导致病毒的基因缺失株无法稳定复制或丧失病毒的免疫原性。
因此，发明人首先在上述实施例1获得的GZ2018毒株的基础上，构建了该毒株的多种I177L基因片段(包括MT496893中第174513-174713(共计201bp，命名为I177L-A，其核苷酸序列如SEQ ID NO:11所示)、第174513-175043(共计531bp，命名为I177L-B，其核苷酸序列如SEQ ID NO:12所示)、第174516-174599(共计84bp，命名为I177L-C，其核苷酸序列如SEQ ID NO:13所示)、第174600-174710(共计111bp，命名为I177L-D，其核苷酸序列如SEQ ID NO:14所示)缺失的基因缺失株，以从中选择确定合适的I177L基因缺失片段，而不会影响到构建的基因缺失株的繁殖稳定性。具体操作包括以下步骤。
2.1、同源重组载体的构建
2.1.1、缺失I177L-A的同源重组载体的构建：利用同源重组的方法，将待缺失部分基因左右同源臂即I177L-A的左同源臂(长度为1200bp左右)和I177L-A的右同源臂(长度为1200bp左右)，与P72启动子基因序列(其核苷酸序列如SEQ ID NO:1所示)和EBFP基因序列(蓝色荧光蛋白表达基因，其核苷酸序列如SEQ ID NO:2所示)合成同源臂，命名为I177L-A-P72-EBFP，将其定向克隆至pBluescript II KS载体(NTCC商购)，获得缺失I177L-A的同源重组转移载体，命名为pBlue-LR-ΔI177L-A-EBFP；
2.1.2、利用同步骤2.1.1的方法还分别构建了缺失I177L-B、I177L-C和I177L-D的同源重组转移载体，分别命名为pBlue-LR-ΔI177L-B-EBFP、pBlue-LR-ΔI177L-C-EBFP和pBlue-LR-ΔI177L-D-EBFP。
2.2、基因缺失重组病毒的构建
用ASFV GZ2018野生毒株感染原代PAM细胞，将步骤2.1构建的同源重组转移载体 pBlue-LR-ΔI177L-A-EBFP、pBlue-LR-ΔI177L-B-EBFP、pBlue-LR-ΔI177L-C-EBFP和pBlue-LR-ΔI177L-D-EBFP分别用TurboFectin8.0转染试剂(购自美国Origene公司)，转染被野生毒株感染的原代PAM细胞，于37℃培养48h后(培养基为含10％FBS的PRMI1640培养基)，分别于显微镜下观察是否有蓝色荧光信号。
结果如图1所示，其中A幅表示转染pBlue-LR-ΔI177L-D-EBFP的原代PAM细胞的荧光照片(有大量的蓝色荧光信号)，B幅表示转染pBlue-LR-ΔI177L-A-EBFP和pBlue-LR-ΔI177L-B-EBFP的原代PAM细胞的荧光照片(没有观察到蓝色荧光信号)，C幅表示转染pBlue-LR-ΔI177L-C-EBFP的原代PAM细胞的荧光照片(仅观察到极少量的蓝色荧光信号)，可见不同I177L基因片段的缺失对病毒复制的影响不同，其中I177L-D片段的缺失不会影响病毒的复制，而I177L-A和I177L-B的缺失会导致病毒无法复制，I177L-C片段的缺失会严重影响病毒的复制能力，而不能稳定增殖。因此，本发明确定缺失I177L-D基因片段，即ASFV GZ2018毒株的MT496893中第174600-174710位核苷酸(共计111bp)，该片段的缺失不会影响构建的基因缺失重组病毒的复制能力，将构建获得的基因缺失重组病毒命名为rASFV GZ2018ΔI177L-D。
实施例3：缺失MGF360(12L,13L,14L)、CD2V和I177L-D基因的重组病毒的构建
对于非洲猪瘟病毒毒力基因的敲除，往往需要考虑到对猪的免疫应答和保护性效果，又要兼顾致病性和安全性能。虽然毒力基因MGF360(12L,13L,14L)的敲除(上述文献1)，或者单独的I177L基因特定片段的敲除(上述文献2)已经被证实均能获得减毒的非洲猪瘟病毒疫苗株，并具有良好的免疫保护效果。然而多种毒力基因的缺失可能会造成的病毒滴度低，也会使致弱毒株降低免疫原性或保护效果减弱，甚至也会影响基因缺失重组病毒的复制能力，而不能拯救病毒。基于此，发明人尝试从ASFV GZ2018毒株出发，构建其同时缺失MGF360(12L,13L,14L)、CD2V和I177L-D基因的重组病毒，并验证是否能够获得稳定复制的基因缺失重组病毒，具体包括以下步骤。
3.1、同源重组载体的构建
3.1.1、缺失MGF360(12L,13L,14L)的同源重组转移载体的构建：利用同源重组的方法，将待缺失部分基因左右同源臂即MGF360(12L,13L,14L)的左同源臂和MGF360(12L,13L,14L)的右同源臂，与P72启动子基因序列和mCherry基因序列(红色荧光蛋白表达基因，其核苷酸序列如SEQ ID NO:3所示)合成同源臂，命名为MGF360-P72-mCherry，将其定向克隆至pBluescript II KS载体，获得缺失MGF360(12L,13L,14L)基因的同源重组转移载体，名命名为pBlue-LR-ΔMGF360-P72-mCherry(全长3301bp)，图2中泳道1-3示出了该重组转移载体的鉴定凝胶电泳图(其中M表示 DL5000 Marker、-表示阴性对照，+表示空载体pBluescript II KS对照)；
3.1.2、缺失CD2V的同源重组转移载体的构建：利用同源重组的方法，将待缺失部分基因左右同源臂即CD2V的左同源臂和CD2V的右同源臂，与EGFP基因序列(绿色荧光蛋白表达基因，其核苷酸序列如SEQ ID NO:4所示)合成同源臂，命名为CD2V-EGFP，将其定向克隆至pBluescript II KS载体，获得缺失CD2V基因的同源重组转移载体，名命名为pBlue-LR-ΔCD2V-EGFP(全长2871bp)，图2中泳道4-6示出了该重组转移载体的鉴定凝胶电泳图；
3.1.3、缺失I177L-D的同源重组转移载体的构建：为实施例2中构建获得的同源重组转移载体pBlue-LR-ΔI177L-D-EBFP(全长3310bp)，图2中泳道7-9示出了该重组转移载体的鉴定凝胶电泳图。
3.2、重组病毒的构建
3.2.1、用ASFV GZ2018野生毒株感染原代PAM细胞，将步骤3.1.1构建的同源重组转移载体pBlue-LR-ΔMGF360-P72-mCherry用TurboFectin8.0转染试剂转染被野生毒株感染的原代PAM细胞，于37℃培养48h后，显微镜下可见存在大量的红色荧光斑点，结果如图3所示，挑取带有红色荧光的细胞至新鲜的原代PAM细胞中，完成一轮纯化，此为P1轮病毒；待P1轮病毒感染细胞扩散至荧光簇，重复上述步骤再次纯化，纯化10轮后，收集荧光细胞，冻融三次，得到表达红色荧光蛋白的MGF360(12L,13L,14L)基因缺失的非洲猪瘟重组病毒，命名为rASFV GZ2018ΔMGF360，其相对于ASFV GZ2018野生毒株缺失了MGF360(12L,13L,14L)的完整阅读框，缺失MGF360(12L,13L,14L)基因的重组病毒的PCR鉴定(鉴定MGF360(12L,13L,14L)基因缺失使用的引物为：5’-aacatgcggtacacacgatg-3’(SEQ ID NO:5)和5’-acggccagcaacaaaagttt-3’(SEQ ID NO:6))结果如图4中泳道1-3所示，其中M表示DL5000 Marker，泳道1表示阳性对照(ASFV GZ2018野生毒株)，泳道2表示重组病毒rASFV GZ2018ΔMGF360，泳道3表示阴性对照。
3.2.2、用ASFV GZ2018野生毒株感染原代PAM细胞，将步骤3.1.2构建的pBlue-LR-ΔCD2V-EGFP按照同上述步骤3.2.1同样的方法转染PAM细胞，在于37℃培养48h后，显微镜下可见存在大量的绿色荧光斑点，结果如图5所示。按照步骤3.2.1的方法重复挑取带有绿色荧光的细胞至新鲜的原代PAM细胞中，最终得到表达绿色荧光蛋白的CD2V基因缺失的非洲猪瘟重组病毒，命名为rASFV GZ2018ΔCD2V，其相对于ASFV GZ2018野生毒株缺失了CD2V的完整阅读框。
3.2.3、用步骤3.2.1获得的rASFV GZ2018ΔMGF360感染原代PAM细胞，将步骤3.1.2 构建的pBlue-LR-ΔCD2V-EGFP按照同上述步骤3.2.1同样的方法转染PAM细胞，并在显微镜下重复挑取红、绿双荧光斑点进行纯化，最终得到表达红、绿双荧光蛋白的MGF360(12L,13L,14L)和CD2V基因都缺失的非洲猪瘟重组病毒，命名为rASFV GZ2018ΔMGF360/ΔCD2V，其相对于ASFV GZ2018野生毒株缺失了MGF360(12L,13L,14L)和CD2V的完整阅读框，PCR鉴定(鉴定CD2V基因缺失使用的引物为：5’-accgcactaggaaaaacggttg-3’(SEQ ID NO:7)和5’-agttggtttgttctcgcagc-3’(SEQ ID NO:8))结果如图4中泳道4-6所示，其中泳道4表示阳性对照(即rASFV GZ2018ΔMGF360重组病毒)，泳道5表示重组病毒rASFV GZ2018ΔMGF360/ΔCD2V，泳道6表示阴性对照。
3.2.4、用步骤3.2.2获得的rASFV GZ2018ΔCD2V感染原代PAM细胞，将步骤3.1.3构建的pBlue-LR-ΔI177L-D-EBFP按照同上述步骤3.2.1同样的方法转染PAM细胞，并在显微镜下重复挑取蓝、绿双荧光斑点进行纯化，最终得到表达蓝、绿双荧光蛋白的I177L-D和CD2V基因都缺失的非洲猪瘟重组病毒，命名为rASFV GZ2018ΔCD2V/ΔI177L-D，其相对于ASFV GZ2018野生毒株缺失了CD2V和I177L-D的阅读框。
3.2.5、用步骤3.2.3获得的rASFV GZ2018ΔMGF360/ΔCD2V感染原代PAM细胞，将步骤3.1.3构建的pBlue-LR-ΔI177L-D-EBFP按照同上述步骤3.2.1同样的方法转染PAM细胞，并在显微镜下重复挑取红、绿、蓝三荧光斑点进行纯化，最终得到表达红、绿、蓝三荧光蛋白的MGF360(12L,13L,14L)、CD2V和I177L-D基因都缺失的非洲猪瘟重组病毒，命名为rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D，其相对于ASFV GZ2018野生毒株缺失了MGF360(12L,13L,14L)、CD2V和I177L-D的阅读框，PCR鉴定(鉴定I177L-D基因缺失使用的引物为：5’-gtgggccccttaagatcaca-3’(SEQ ID NO:9)和5’-ccactctgatactccccagc-3’(SEQ ID NO:10))结果如图4中泳道7-9所示，其中泳道7表示重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D，泳道8表示阳性对照(即rASFV GZ2018ΔMGF360/ΔCD2V重组病毒)，泳道9表示阴性对照。
经测序结果表明，该实施例3成功构建了同时缺失MGF360(12L,13L,14L)、CD2V和I177L-D的重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D，并且该重组病毒能够在原代PAM细胞中稳定复制繁殖，在感染PAM细胞时能够表达红、绿、蓝三色荧光，不会出现由于多种毒力基因的缺失而导致重组病毒无法复制繁殖的问题。
实施例4：重组病毒的滴度测定
该实施例对上述实施例2构建的重组病毒rASFV GZ2018ΔI177L-D，上述实施例3构 建的重组病毒rASFV GZ2018ΔCD2V/ΔI177L-D以及重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的滴度进行测定，并与野生毒株ASFV GZ2018的滴度进行比较。非洲猪瘟病毒的滴度测定分别采用半数细胞感染剂量TCID 50和半数血球吸附量HAD 50两种方法进行操作，其中：
TCID 50滴定按照下面步骤进行：将ASFV以无血清1640培养液再作10倍连续稀释，接种培养于96孔培养板、密度约90-100％的PAM细胞，每个稀释度接种8个孔，100μl/孔，于37℃、5％浓度CO 2条件下培养，观察3-7天，根据细胞病变或荧光变化和Reed and Muench方法计算半数细胞感染量(TCID 50)。
HAD 50试验操作按照《非洲猪瘟病毒红血球吸附试验操作规程》进行操作，并作适当调整：在96孔细胞培养板中接种原代PAM细胞，将待检样品进行10倍梯度稀释，20μl/孔，病毒感染可根据红细胞在感染细胞周围聚集形成的玫瑰花环进行判定，观察7天，根据Reed and Muench方法计算半数血球吸附剂量(HAD 50)。
具体测定操作包括以下步骤。
(1)将猪原代PAM细胞铺入6孔板中，长成单层后，分别接种0.1MOI病毒液(ASFV GZ2018、rASFV GZ2018ΔI177L-D、rASFV GZ2018ΔCD2V/ΔI177L-D或rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D)，37℃孵育2h，分别于感染后0h、6h、12h、18h、24h、30h、36h、48h、72h收获扩增病毒，反复冻融3次，高速离心，取上清，分别获得培养病毒液；
(2)分别将上述步骤(1)获得的培养病毒液按照10倍梯度进行稀释，感染已铺在96孔板中形成单层的猪原代PAM细胞，于感染后72h测定HAD 50；基因缺失重组病毒也可直接利用荧光显微镜观察，按照Reed-Muench法计算病毒的滴度，以TCID 50/mL为单位。
结果显示：在所测定的72h时间内，三株基因缺失重组病毒的平均滴度均低于野生毒株ASFV GZ2018，其中在感染后48h时，ASFV GZ2018、rASFV GZ2018ΔI177L-D、rASFV GZ2018ΔCD2V/ΔI177L-D及rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的病毒滴度分别为10 7.20TCID 50/mL、10 4.30TCID 50/mL、10 4.28TCID 50/mL、10 4.25TCID 50/mL；在感染后72h时，ASFV GZ2018、rASFV GZ2018ΔI177L-D、rASFV GZ2018ΔCD2V/ΔI177L-D及rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的病毒滴度分别为10 7.50TCID 50/mL、10 5.80TCID 50/mL、10 5.65TCID 50/mL、10 5.25TCID 50/mL。可见，在感染PAM细胞后，基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的滴度明显低于野生毒株ASFV GZ2018的滴度(前者比后者低100倍至1000倍)，也明显低于 文献1公开的SY18ΔMC组病毒(缺失MGF360(12L,13L,14L)和CD2V)的滴度(感染后48h的平均滴度为8.472×10 6TCID 50/mL)，并且与文献2公开的只缺失I177L基因片段的基因缺失株ASFV-G-ΔI177L的毒力相当，并没有因为缺失更多的毒力基因而导致基因缺失株rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的病毒滴度过低和免疫原性的过渡致弱。
因此本发明构建获得的基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D是一种相对于野生毒株明显减毒的非洲猪瘟病毒基因缺失重组病毒，并且仍保持相对较高的病毒滴度和较高的免疫原性。理论上，在不降低免疫原性的情况下，毒力基因缺失越多，病毒毒力越弱，其安全风险就越低，因此本发明提供的同时缺失MGF360(12L,13L,14L)、CD2V和I177L-D基因的重组病毒相对于野生毒株、文献1和文献2公开的基因缺失株将具有更高的安全性，并且由于I177L在基因组中为单一基因，不会轻易发生自身突变和同源重组缺失或回补缺失部位的现象，也可以避免潜在的毒力返强安全风险。
实施例5：基因缺失株rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的毒力检测
该实施例对上述实施例3获得的基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的毒力进行检测，以对其安全性进行评价，具体包括以下步骤。
(1)选取20头健康仔猪(非洲猪瘟病毒抗原、抗体均阴性)，购自内蒙古包头市草原立新猪场，随机分为4组(A、B、C和D组)；
(2)分别选用10 3TCID 50/头低感染剂量和10 5TCID 50/头高感染剂量的rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D对A和B组仔猪进行肌肉注射；同时采用感染剂量为10 3TCID 50/头的野生毒株ASFV GZ2018对C组仔猪进行肌肉注射，作为对照；还采用1640培养基(2ml)作为阴性对照肌肉注射D组仔猪；试验方案如下表1所示；
(3)接种后每日对各组仔猪测定采食量、体温、体重变化情况，并记录仔猪存活情况，直至接种后28天；在观察期内，每5天对各组仔猪采集猪外周血、唾液、粪便和泪腺(若仔猪死亡，则停止对该死亡仔猪进行采集)，以检测仔猪血液中病毒含量(HAD 50)和唾液、粪便以及泪腺等部位是否存在排毒情况。
表1：基因缺失株rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D的毒力检测试验方案
Figure PCTCN2022086526-appb-000001
Figure PCTCN2022086526-appb-000002
结果显示：C组仔猪经肌肉注射接种10 3.0TCID 50/头剂量ASFV GZ2018毒株后，所有仔猪均在接种3日后发生高热，高达42℃以上，精神沉郁，4～6天左右开始死亡，并在10天内全部死亡。剖检可见淋巴结、脾脏、肾脏等部位严重出血，病毒血症一直持续到猪只死亡。而使用rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D接种后，无论是低剂量(10 3.0TCID 50/头)还是高剂量(10 5.0TCID 50/头)接种后，所有仔猪的平均体温均未超过40.3℃，后期亦未见体温异常，精神状态正常，采食量未见异常，体重增加明显。另外，观察期内所有仔猪无病毒血症发生，粪便、唾液、泪腺等部位无排毒现象，相对于文献2公开的基因缺失株ASFV-G-ΔI177L，接种本发明提供的基因缺失株rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D后，检测的仔猪血液中病毒含量一直处于更低水平，这与实施例4中重组病毒的滴度检测结果一致。
该实施例结果表明，本发明构建的减毒的基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D对仔猪确实具有良好的安全性。
实施例6：基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D作为疫苗株对仔猪的免疫保护效果评价
该实施例利用基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D作为疫苗株对仔猪进行免疫，以通过抗体检测和攻毒试验评价其免疫保护效果，具体包括以下步骤。
(1)选取3～4周龄健康仔猪20头，随机分成3组(a、b、c和d组)，每组5头；a组仔猪用基因缺失株rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D于颈部肌肉注射，10 3.0TCID 50/头，2周后同样剂量加强免疫一次；b组仔猪用基因缺失株rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D于颈部肌肉注射，10 5.0TCID/头，2周后同样剂量加强免疫一次；c组和d组为阴性对照，同样方法注射生理盐水2ml；三组分别隔离饲养。试验方案如下表2所示。
(2)免疫后抗体检测：免疫后分别于3日、5日、7日、14日、21日和28日从各组仔猪的前腔静脉采血，对非洲猪瘟病毒抗体进行检测(英吉纳抗体试剂盒)，评价其免疫效果。
(3)攻毒保护试验：接种后第28日，从各组仔猪采血结束后，a组、b组和c组仔猪颈部肌肉接种ASFV GZ2018(50HAD 50/头)进行攻毒保护试验，d组仔猪注射生理盐水2ml。
表2：免疫保护效果评价试验方案
Figure PCTCN2022086526-appb-000003
抗体检测结果显示：a组和b组仔猪于免疫后3日、5日和7日均未产生特异性抗体；z组仔猪于免疫后14日、21日及28日后抗体阳性率分别为40％、80％和100％；b组仔猪于免疫后14日、21日和28日抗体阳性率均为100％。c组和d组仔猪在观察期内均未产生特异性抗体。在观察期内，a组和b组仔猪的体温均在正常温度范围之内，未见任何异常临床表现，无病毒血症发生，粪便、唾液、泪腺等部位无排毒。剖检可见所有免疫仔猪未发现明显病理变化。
免疫攻毒保护试验结果显示：a组和b组仔猪免疫攻毒后的保护率均为100％，而c组5头仔猪全部于攻毒后12日内死亡，d组仔猪表现正常。结果如下表3所示。
表3：免疫攻毒试验结果
Figure PCTCN2022086526-appb-000004
综上所述，可见当将本发明构建的基因缺失重组病毒rASFV GZ2018ΔMGF360/ΔCD2V/ΔI177L-D作为疫苗株按照首次免疫后14日加强免疫一次的方案接种仔猪后，不论是低剂量组(10 3.0TCID 50/头)还是高剂量组(10 5.0TCID 50/头)均能产生较好的免疫应答，提供100％的攻毒保护效果。其中10 3.0TCID 50/头低剂量组在首免后14日二免前抗体转阳率为40％，二免后7日和14日抗体阳性率持续上升，攻毒前抗体阳性率达到100％；10 5.0TCID 50/头高剂量组在首免后14日二免前即可达到100％抗体阳性率，并且所有仔猪体温均在正常范围之内，未见任何异常临床表现，无病毒血症发生，粪便、唾液、泪腺等部位无排毒，接种后血液病毒含量一直处于较低水平。表明野生毒株ASFV GZ2018缺失MGF360(12L,13L,14L)、CD2v和I177L-D后仍然保留较高的免疫原性并且免疫健康易感猪后可提供有效的攻毒保护效果，该基因缺失株可以 提供安全、有效的免疫保护作用，因此可以作为一种候选的非洲猪瘟病毒疫苗株。在作为疫苗株制备减毒活疫苗时，可以将本发明构建的基因缺失的减毒非洲猪瘟病毒毒株接种原代PAM细胞，扩大培养，收获病毒液并测定病毒滴度，可以在浓缩后选择病毒含量≥10 7.0TCID 50/ml的病毒液直接或与配合佐剂(例如纳米佐剂、白介素或干扰素)制成疫苗，制备得到的疫苗中的病毒含量可高达10 7.0TCID 50/ml以上。
此处描述的实施例只用于说明(作为例证)，技术人员所做的各种修改或变更也应包括在专利申请的实质范围内。
工业应用性
本发明提供了一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用，该基因缺失的减毒非洲猪瘟病毒毒株通过同源重组方法构建，相对于亲本毒株明显减毒，且不会影响基因缺失病毒株的稳定复制和免疫原性，可以作为一种具有良好安全性和免疫保护效果的候选疫苗株，适于工业应用。

Claims (9)

  1. 一种基因缺失的减毒非洲猪瘟病毒毒株，其在II型非洲猪瘟病毒的基因组中缺失以下基因或片段：
    CD2V基因、MGF360(12L,13L,14L)和I177L基因片段，其中所述MGF360(12L,13L,14L)表示MGF360-12L基因、MGF360-13L基因和MGF360-14L基因，所述I177L基因片段的核苷酸序列如SEQ ID NO:14所示。
  2. 根据权利要求1所述的减毒非洲猪瘟病毒毒株，其中所述II型非洲猪瘟病毒包括但不限于ASFV-SY18、Georgia 2008/1、Pig/HLJ/2018、Georgia 2007/1、ASFV GZ2018。
  3. 权利要求1或2所述的减毒非洲猪瘟病毒毒株的构建方法，其包括以下步骤：通过基因工程手段将亲本II型非洲猪瘟病毒的CD2V基因、MGF360(12L,13L,14L)基因和I177L基因片段缺失，其中所述I177L基因片段的核苷酸序列如SEQ ID NO:14所示。
  4. 根据权利要求3所述的构建方法，其中所述基因工程手段为同源重组技术，具体包括以下步骤：
    S1)将MGF360(12L,13L,14L)基因的左右同源臂和第一筛选表达盒克隆至pBluescript II KS载体，获得第一同源重组转移载体；
    S2)将CD2V基因的左右同源臂和第二筛选表达盒克隆至pBluescript II KS载体，获得第二同源重组转移载体；
    S3)将I177L基因片段的左右同源臂和第三筛选表达盒克隆至pBluescript II KS载体，获得第三同源重组转移载体；
    S4)将步骤S1)的第一同源重组转移载体用转染试剂转染至使用所述亲本II型非洲猪瘟病毒感染的原代PAM细胞中，通过所述第一筛选表达盒筛选获得缺失MGF360(12L,13L,14L)基因的第一重组病毒；
    S5)将步骤S2)的第二同源重组转移载体用转染试剂转染至使用步骤S4)获得的第一重组病毒感染的原代PAM细胞中，通过所述第一筛选表达盒和第二筛选表达盒筛选获得同时缺失MGF360(12L,13L,14L)基因和CD2V基因的第二重组病毒；
    S6)将步骤S3)的第三同源重组转移载体用转染试剂转染至使用步骤S5)获得的第二重组病毒感染的原代PAM细胞中，通过所述第一筛选表达盒、第二筛选表达盒和第三筛选表达盒筛选获得同时缺失MGF360(12L,13L,14L)基因、CD2V基因和I177L基因片段的第三重组病毒，作为减毒非洲猪瘟病毒毒株。
  5. 根据权利要求4所述的构建方法，其中所述第一筛选表达盒、第二筛选表达盒和第三筛选表达盒为不同的筛选表达盒，均选自mCherry、EGFP和EBFP表达盒。
  6. 一种非洲猪瘟病毒减毒活疫苗，其由权利要求1或2所述的基因缺失的减毒非洲猪瘟病毒毒株制备获得。
  7. 根据权利要求6所述的非洲猪瘟病毒减毒活疫苗，其中的减毒非洲猪瘟病毒的病毒含量≥10 7.0TCID 50/ml。
  8. 权利要求6或7所述的非洲猪瘟病毒减毒活疫苗的制备方法，其包括以下步骤：
    T1)将权利要求1或2所述的基因缺失的减毒非洲猪瘟病毒毒株接种原代PAM细胞，扩大培养，收获病毒液；
    T2)以病毒含量≥10 7.0TCID 50/ml的病毒液直接或配合佐剂制成疫苗。
  9. 根据权利要求8所述的制备方法，其中所述佐剂包括但不限于纳米佐剂、白介素和干扰素。
PCT/CN2022/086526 2021-04-13 2022-04-13 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用 WO2022218325A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110394476.8A CN113122511B (zh) 2021-04-13 2021-04-13 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用
CN202110394476.8 2021-04-13

Publications (1)

Publication Number Publication Date
WO2022218325A1 true WO2022218325A1 (zh) 2022-10-20

Family

ID=76776038

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/086526 WO2022218325A1 (zh) 2021-04-13 2022-04-13 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用

Country Status (2)

Country Link
CN (1) CN113122511B (zh)
WO (1) WO2022218325A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116492456A (zh) * 2023-03-31 2023-07-28 中国人民解放军军事科学院军事医学研究院 非洲猪瘟病毒d129l基因及其在制备复制缺陷型非洲猪瘟疫苗中的应用
CN117535447A (zh) * 2023-11-09 2024-02-09 中国农业科学院北京畜牧兽医研究所 一种鉴别非洲猪瘟病毒的多重荧光定量pcr引物、探针及检测方法
CN117701509A (zh) * 2023-11-20 2024-03-15 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 一种非洲猪瘟病毒传代致弱毒株与基于该毒株的疫苗

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113122511B (zh) * 2021-04-13 2021-11-19 金宇保灵生物药品有限公司 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用
CN114134253A (zh) * 2021-11-10 2022-03-04 佛山科学技术学院 鉴别非洲猪瘟野毒株和基因缺失株的荧光定量检测引物及试剂盒
CN115851626B (zh) * 2022-07-13 2023-07-25 金宇保灵生物药品有限公司 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110551695A (zh) * 2019-08-14 2019-12-10 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 非洲猪瘟病毒四基因缺失弱毒株及其应用
CN112063634A (zh) * 2020-05-01 2020-12-11 中国农业科学院兰州兽医研究所 一种基因缺失的减毒非洲猪瘟病毒株及应用
CN112063633A (zh) * 2020-03-29 2020-12-11 中国农业科学院兰州兽医研究所 一种天然免疫抑制基因缺失的减毒非洲猪瘟病毒株及应用
CN112063592A (zh) * 2020-07-10 2020-12-11 中国农业科学院兰州兽医研究所 非洲猪瘟多基因联合缺失减毒株的构建及作为疫苗的应用
US20210085776A1 (en) * 2019-09-24 2021-03-25 The United States Of America, As Represented By The Secretary Of Agriculture Development of a novel live attenuated african swine fever vaccine based in the deletion of gene i177l
CN112626101A (zh) * 2021-01-06 2021-04-09 佛山科学技术学院 非洲猪瘟病毒i177l基因重组质粒、表达载体及制备方法
CN113122511A (zh) * 2021-04-13 2021-07-16 金宇保灵生物药品有限公司 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022525920A (ja) * 2019-03-27 2022-05-20 ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハー アフリカ豚熱ウイルスペプチドおよびタンパク質を含む免疫原性組成物およびワクチンならびにその使用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110551695A (zh) * 2019-08-14 2019-12-10 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 非洲猪瘟病毒四基因缺失弱毒株及其应用
US20210085776A1 (en) * 2019-09-24 2021-03-25 The United States Of America, As Represented By The Secretary Of Agriculture Development of a novel live attenuated african swine fever vaccine based in the deletion of gene i177l
CN112063633A (zh) * 2020-03-29 2020-12-11 中国农业科学院兰州兽医研究所 一种天然免疫抑制基因缺失的减毒非洲猪瘟病毒株及应用
CN112063634A (zh) * 2020-05-01 2020-12-11 中国农业科学院兰州兽医研究所 一种基因缺失的减毒非洲猪瘟病毒株及应用
CN112063592A (zh) * 2020-07-10 2020-12-11 中国农业科学院兰州兽医研究所 非洲猪瘟多基因联合缺失减毒株的构建及作为疫苗的应用
CN112626101A (zh) * 2021-01-06 2021-04-09 佛山科学技术学院 非洲猪瘟病毒i177l基因重组质粒、表达载体及制备方法
CN113122511A (zh) * 2021-04-13 2021-07-16 金宇保灵生物药品有限公司 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN YANXING, CAO CHENFU, SHI WEIJUN, HUANG CHAOHUA, ZENG SHAOLING, SUN JIE, WU JIANG, HUA QUNYI: "Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 280, 1 June 2020 (2020-06-01), NL , pages 113875 - 5, XP055976952, ISSN: 0166-0934, DOI: 10.1016/j.jviromet.2020.113875 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116492456A (zh) * 2023-03-31 2023-07-28 中国人民解放军军事科学院军事医学研究院 非洲猪瘟病毒d129l基因及其在制备复制缺陷型非洲猪瘟疫苗中的应用
CN116492456B (zh) * 2023-03-31 2024-04-23 中国人民解放军军事科学院军事医学研究院 非洲猪瘟病毒d129l基因及其在制备复制缺陷型非洲猪瘟疫苗中的应用
CN117535447A (zh) * 2023-11-09 2024-02-09 中国农业科学院北京畜牧兽医研究所 一种鉴别非洲猪瘟病毒的多重荧光定量pcr引物、探针及检测方法
CN117701509A (zh) * 2023-11-20 2024-03-15 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 一种非洲猪瘟病毒传代致弱毒株与基于该毒株的疫苗
CN117701509B (zh) * 2023-11-20 2024-05-28 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 一种非洲猪瘟病毒传代致弱毒株与基于该毒株的疫苗

Also Published As

Publication number Publication date
CN113122511B (zh) 2021-11-19
CN113122511A (zh) 2021-07-16

Similar Documents

Publication Publication Date Title
WO2022218325A1 (zh) 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用
JP6845266B2 (ja) 多価組換型鳥ヘルペスウイルス及び鳥類を免疫化するためのワクチン
Liu et al. A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus infection in ducks
CN111218459A (zh) 一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗
JP2016531545A (ja) 豚ヘルペスウイルス遺伝子欠失株、ワクチン組成物及びその製造方法と応用
CN111019910B (zh) F基因型腮腺炎病毒减毒株及构建方法和应用
US11033617B2 (en) Duck hepatitis A virus type 3 mutant CH-P60-117C and construction thereof
CN112063592A (zh) 非洲猪瘟多基因联合缺失减毒株的构建及作为疫苗的应用
WO2022007742A1 (zh) 一种重组的伪狂犬病病毒及其疫苗组合物
Zhang et al. Protection conferred by a recombinant Marek’s disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken
CN109136198B (zh) 一种表达鸡传染性贫血病毒vp1、vp2基因重组鸡痘病毒活载体疫苗
CN111748563A (zh) 非洲猪瘟基因缺失弱毒株的构建及其作为疫苗的应用
WO2003064595A2 (en) Recombinant herpesvirus of turkeys and use thereof
UA123682C2 (uk) Вірус качиного ентериту та його застосування
CN115851623A (zh) 非洲猪瘟mgf505-2r基因缺失减毒株的构建及作为疫苗的应用
US20220204568A1 (en) Method for rapid preparation of epidemic infectious bronchitis vaccine
CN113817753B (zh) 表达SARS-CoV-2纤突蛋白或其变异体SΔ21的假型化VSV病毒构建和应用
CN114015660B (zh) 缺失十个基因的减毒非洲猪瘟病毒株的构建及其作为疫苗的应用
JP2003503039A (ja) 弱毒化したワクチンの操作のための方法および組成物
WO2024012483A1 (zh) 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用
Williams et al. A recombinant bovine herpesvirus-4 vectored vaccine delivered via intranasal nebulization elicits viral neutralizing antibody titers in cattle
Jia et al. Construction and immune efficacy of a recombinant turkey herpesvirus vaccine strain expressing fusion protein of genotype VII Newcastle disease virus
CN116286677B (zh) 一种非洲猪瘟病毒毒株及其应用
JP4474164B2 (ja) ワクチン製造のための連続継代性細胞系
Li et al. Development and characterization of Rift Valley fever virus-like particles

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22787547

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22787547

Country of ref document: EP

Kind code of ref document: A1