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CN-118421572-B - Construction of attenuated African swine fever virus strain with twenty-four deleted genes and application of attenuated African swine fever virus strain as vaccine

CN118421572BCN 118421572 BCN118421572 BCN 118421572BCN-118421572-B

Abstract

The invention particularly relates to an attenuated African swine fever virus strain with twenty-four genes deleted and application of the strain as a vaccine. The invention combines and deletes KP177R, L/GS-2018 virus L, L-L, ASFV-G-ACD-00090, ASFV-G-ACD-00120, 285L, ASFV-G-ACD-00160 and 24 genes of MGF-360, MGF-110, MGF-300, MGF-100 and the like in ASFV-CN/GS-2018 virus to obtain an attenuated African swine fever vaccine strain, which completely weakens pigs after immunization, is healthy for immunized pigs, detects the existence of high-level protective antibodies in pig blood after immunization, can provide 100% immune protection for ASFV CN/GS/2018 virulent strain challenge, can be used as a safe and effective African swine fever candidate vaccine, and has great social value.

Inventors

  • ZHENG HAIXUE
  • ZHU ZIXIANG
  • XU FAN
  • DANG WEN
  • DING MINGYANG
  • LI TAO
  • DU YU
  • LIU HUANAN
  • SHI ZHENGWANG
  • TIAN HONG

Assignees

  • 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心)

Dates

Publication Date
20260508
Application Date
20240511

Claims (7)

  1. 1. The application of preparing an attenuated African swine fever virus strain by combining deletion of gene fragments in an African swine fever virus strain ASFV CN/GS 2018 is characterized in that the gene fragments are 852-1403, 1613-1933, 2018-3106, 3247-3780, 3917-4162, 4263-4421, 4580-5650, 6096-6686, 6685-6798, 6866-7180, 7277-7651, 7797-7954, 7965-8339, 8528-9145, 9352-9765, 10080-64, 10106-10234, 10493-10876, 11094-11468, 19732-20538, 21365-93847, 21957-1809, 180179517-7685, and the strain are in the African swine fever virus strain ASFV CN/GS 2018, and the strain is a typical strain preserved in the African swine fever virus culture center No. 25, and the strain is a strain with the preservation number of 202096.
  2. 2. Use of a gene fragment deleted in a strain of african swine fever virus type II ASFV CN/GS 2018 for the preparation of an african swine fever vaccine, characterized in that the gene fragment deleted is from positions 852-1403, 1613-1933, 2018-3106, 3247-3780, 3917-4162, 4263-4421, 4580-5650, 6096-6686, 6685-6798, 6866-7180, 7277-7651, 7797-7954, 7965-8339, 10328-9145, 9352-9765, 10106-10234, 10493-10876, 11094-31068, 19732-20538, 65-21847, 219517-219, 1801799309-617 and 617 of the full length sequence of the strain of african swine fever virus type II ASFV CN/GS 2018; the African swine fever virus strain II ASFV CN/GS 2018 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of V202096.
  3. 3. The gene deletion attenuated African swine fever virus strain is a gene fragment deleted type II African swine fever virus strain ASFV CN/GS 2018, and the gene fragment is 852-1403, 1613-1933, 2018-3106, 3247-3780, 3997-4162, 4263-4421, 4580-5650, 6096-6686, 6685-6798, 6866-7190, 7277-7685, 7797-7954, 7965-8339, 8528-9145, 9352-9765, 10080-10364, 10106-10234, 10493-10876, 11094-68, 19732-20538, 21365-212121219917-179906-1149, and 11499309-11499309.
  4. 4. An african swine fever vaccine comprising the gene-deleted attenuated african swine fever virus strain of claim 3.
  5. 5. A method for preparing the gene deletion attenuated african swine fever virus strain of claim 3, wherein the method is to delete positions 852-1403, 1613-1933, 2018-3106, 3247-3780, 3917-4162, 4263-4421, 4580-5650, 6096-6686, 6685-6798, 6866-7190, 7277-7651, 7797-7954, 7965-8339, 8528-9145, 9352-9765, 10080-64, 10106-10234, 10493-10876, 11094-11468, 19732-20538, 21365-21847, 21937-229, 9519-179917-180309 and 180309-180309 of the full length sequence of the original african swine fever virus strain ASFV/GS 2018 by genetic engineering means.
  6. 6. The method of claim 5, wherein the method is a homologous recombination technique.
  7. 7. A method of preparing the gene-deleted attenuated african swine fever virus strain of claim 3, comprising the steps of: (1) Designing 1.0kb of each of upstream and downstream sequences of MGF-100-1R genes as left and right homologous recombination arms, and cloning the left and right homologous recombination arms and BFP gene screening expression cassette gene fragment p72-BFP to a pUC57 vector simultaneously to obtain a recombinant plasmid A00-A-BFP; (2) Designing 1.0kb of each of an upstream sequence of the MGF-100-2L gene and a downstream sequence of the MGF-100-3L gene as a left homologous recombination arm and a right homologous recombination arm gene and a mCherry gene screening expression cassette gene fragment p72-mCherrry are cloned to a pUC57 vector at the same time to obtain a recombinant plasmid A00-BC-RFP; (3) Designing 1.0kb of each of an upstream sequence of the MGF-300-1L gene and a downstream sequence of the MGF-300-4L gene as a left homologous recombination arm and a right homologous recombination arm gene and an eGFP gene screening expression cassette gene fragment p72-eGFP to clone into a pUC57 vector simultaneously to obtain a recombinant plasmid C00-GFP; (4) The recombinant plasmids C00-GFP, A00-BC-RFP and A00-A-BFP are sequentially transfected into BMDM cells of an ASFV original strain, and the recombinant plasmids are continuously passaged to construct and obtain MGF-360-1La genes, MGF-360-1Lb genes, MGF-360-2L genes, KP177R genes, L83L genes, L60L genes, MGF-360-3L genes, MGF-110-1L genes, ASFV-G-ACD-00090 genes, MGF-110-2L genes, MGF-110-3L genes, ASFV-G-ACD-00120 genes, MGF-110-4L genes, MGF-110-5L-6L genes, MGF-110-7L genes, 285L genes, ASFV-G-ACD-00160 genes, MGF-110-8L genes, MGF-100-1R genes, MGF-300-1L genes, MGF-300-2R genes, MGF-300-8L genes, MGF-100-24L genes and ASFV-2-3L genes.

Description

Construction of attenuated African swine fever virus strain with twenty-four deleted genes and application of attenuated African swine fever virus strain as vaccine Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to construction of an attenuated African swine fever virus strain ASFV-delta 24 with twenty-four genes deleted and application of the attenuated African swine fever virus strain ASFV-delta 24 as a vaccine. Background African swine fever (AFRICAN SWINE FEVER, ASF) is a high-morbidity and high-mortality infectious disease caused by African swine fever Virus (AFRICAN SWINE FEVER Virus, ASFV), wild pigs and domestic pigs are susceptible animals, and the high-toxicity strain can cause the mortality of the domestic pigs to be up to 100%. ASFV is the only member of the genus African swine fever virus (ASFARVIRIDAE) and is also the only DNA virus that can be transmitted by arthropods (arbovirus ). ASFV is a double-stranded DNA virus wrapped in a multilayer structure, the diameter is about 200nm, viral nucleic acid at the innermost part of the virion is wrapped by a Core capsid (Core membrane), while the Core capsid is wrapped by an Inner membrane (also called Inner lipid membrane), which is a protein capsid (Capsid) of icosahedral structure outside the Inner membrane, and the outermost layer of the virus is wrapped by a plasma membrane (Plasmama membrane). Wherein p72 is the major capsid forming protein (encoded by the B646L gene, derived from pp220 and pp 62) and represents about 33% of the total capsid protein. The p72 gene sequence is the basis of ASFV genotyping, p72 is also the main antigen for ASFV diagnosis, various detection reagents are developed according to p72 protein information, and the monoclonal antibodies thereof are found to have the capability of neutralizing ASFV. African swine fever causes immeasurable economic loss, and vaccine prevention and control are the most effective means for clearing African swine fever. In the research at home and abroad, the vaccine for African swine fever comprises four major types, namely 1, an inactivated vaccine, which is the safest vaccine for preventing and controlling African swine fever epidemic situation, has no risk of transmitting viruses, has extremely low protection rate and is difficult to meet the prevention and control requirements in production, 2, an attenuated live vaccine, which has extremely high protection rate against parent strains, has simple preparation process and is easy to generate the risk of virus re-strengthening, aggravates the influence of epidemic situation, 3, a subunit vaccine, which removes the genome structure of viruses, ensures the safety of the vaccine, retains important proteins of the viruses, ensures stable expression in pigs, resists the invasion of African swine fever, has more complex preparation process and longer production period, and 4, a genetic engineering vaccine, which can be divided into a DNA vaccine, an RNA vaccine, a gene knockout vaccine and the like, combines the novel genetic engineering technology, reforms ASFV from the aspect of genome to weaken the virulence after deleting certain genes, and simultaneously can induce the immunity of organisms and protect hosts. In the further exploration of the vaccines, no effective commercial vaccines are yet developed. The research is to use genetic engineering technology, and attenuated strains are formed by knocking out virulence genes of ASFV or genes involved in host immunity, so that the toxicity is more stable, the possibility of virulence return is smaller, and the safety is higher. Such vaccines have been reported in a number of papers. Studies have shown that Vivian O' Donnell et al knocked out the B119L and DP96R genes in the 2007 Gregorian ASFV (ASFV-G) genome, constructed ASFV-G- Δ9GL/ΔUK strain that provided 100% protection to swine herds after 14 days of immunization, but that had viremia, and that the protective effects were associated with the presence of ASFV antibodies in serum, independent of cells secreting specific IFN-gamma. The gene I267L plays an important role in ASFV replication, and promotes virus replication by inhibiting RNA Pol-III-RIG-I mediated innate anti-viral immune response, and RanYong et al construct ASFV-delta I267L gene deletion virus (deletion of the gene I267L), and the deletion virus can provide 80% protection rate for parents after animal experiment evaluation. Manuel V Borca et al found that the EP402R gene encodes a transmembrane protein of ASFV and thus participates in immunomodulation of the host, and experiments have also shown that deletion of the EP402R gene does not provide any protection rate. There is also a special gene in the genome of African swine fever, namely a polygene family (Multi-GENES FAMILY, MGF), and 5 classes of MGF are known to be included in ASFV of different genotypes, namely MGF100(1R/2L/3L)、MGF110(1L/2L/3L/4L/5L/6L/7L/8L/9L/10L/1