A gene-deleted attenuated African swine fever virus strain and its construction method and application

An African swine fever virus and gene deletion technology, applied in the field of biological vaccine products, can solve the problems of self-mutation, homologous recombination loss, and strong virulence.

Active Publication Date: 2021-11-19
JINYUBAOLING BIO PHARMA CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the phenomenon that MGF gene fragments such as MGF360-12L, MGF-13L, and MGF-14L are prone to self-mutation and homologous recombination deletion or complementation of the deletion site is very likely to cause the deletion of CD2v using the above-mentioned literature 1. and MGF360 (12L, 13L, 14L) gene vaccine strains to prepare live attenuated vaccines face potential safety risks of virulence reversion
In view of the multifaceted safety problems in the above patent document 1, there is an urgent need for a safer African swine fever virus vaccine product

Method used

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  • A gene-deleted attenuated African swine fever virus strain and its construction method and application
  • A gene-deleted attenuated African swine fever virus strain and its construction method and application
  • A gene-deleted attenuated African swine fever virus strain and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: Isolation and Identification of African Swine Fever Virus Strains

[0035] 1.1. Virus strains

[0036] The wild strain of African swine fever virus used in the embodiment is the Guangdong strain isolated from the wild by the Poultry Disease Teaching and Research Office of the Veterinary College of South China Agricultural University, named ASFV GZ2018, and the specific operations include the following steps (operated in a biosafety level 3 laboratory ): Weigh 0.1g of the spleen tissue of a dead pig that is detected as positive for ASFV infection, put it into a 2.0mL sterile EP tube, add 1mL of sterile PBS solution into the tube, and then put 3 pieces of sterile tissue for grinding Steel balls, put the EP tube into the tissue grinder, and perform vibration grinding. Note that the grinder is pre-cooled to 4°C in advance and placed in a balanced position. After repeated freezing and thawing of the ground tissue fluid for 3 times, filter it with a 0.45 μm filt...

Embodiment 2

[0040] Example 2: Determination of I177L Gene Deletion Fragment

[0041] Although Manuel V.Borca et al. (Development of a highly effective Africanswine 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 Said document 2) It has been proved that the I177L gene segment is deleted in the genome of type II African swine fever virus Georgia2007 / 1 (the Genebank accession number of Georgia 2007 / 1 is NC_044959.1, wherein the I177L gene segment deletion position is No. 174530-174671 (a total of 142bp ) in 112bp; equivalent to 112bp in 174572-174713 in the genome MT496893 of the ASFV GZ2018 strain) can obtain the gene deletion vaccine strain ASFV-G-ΔI177L with good safety and immune protection effect. However, 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 wil...

Embodiment 3

[0049] Embodiment 3: Construction of the recombinant virus of deletion MGF360 (12L, 13L, 14L), CD2V and I177L-D gene

[0050] For the knockout of the African swine fever virus virulence gene, it is often necessary to take into account the immune response and protective effect on pigs, as well as the pathogenicity and safety performance. Although the knockout of the virulence gene MGF360 (12L, 13L, 14L) (above document 1), or the knockout of a specific fragment of the I177L gene alone (above document 2) has been proven to be able to obtain attenuated African swine fever virus vaccine strain, and has a good immune protection effect. However, the deletion of multiple virulence genes may cause low virus titers, reduce the immunogenicity of the attenuated virus strain or weaken the protective effect, and even affect the replication ability of the gene-deleted recombinant virus, but cannot rescue the virus. Based on this, the inventors tried to construct a recombinant virus that si...

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Abstract

The invention discloses a gene-deleted attenuated African swine fever virus strain, a construction method and application thereof, and belongs to the technical field of biological vaccine products. The gene-deleted attenuated African swine fever virus strain constructed by the homologous recombination method of the present invention is based on the type II African swine fever virus genome and simultaneously deletes CD2v, MGF (12L, 13L, 14L) and I177L gene fragments The gene deletion virus strain is obviously attenuated relative to the parental strain, and will not affect the stable replication and immunogenicity of the gene deletion virus strain. After being inoculated into experimental pigs, there will be no obvious rise in body temperature, joint swelling, disease or death of experimental pigs, and no viremia, showing good safety and good immune challenge protection effect. Therefore, the gene-deleted attenuated African swine fever virus strain provided by the present invention can be used as a candidate vaccine strain with good safety and immune protection effect.

Description

technical field [0001] The invention belongs to the technical field of biological vaccine products, in particular to a gene-deleted attenuated African swine fever virus strain and its construction method and application, especially to a simultaneous deletion of CD2v, MGF (12L, 13L, 14L) and I177L genes Attenuated African swine fever virus strain of partial fragments, its construction method and application in vaccine preparation. Background technique [0002] African swine fever virus (ASFV) is the only African virus genus in the African virus family, and it is also the only DNA arbovirus genus. The virus is mainly infected by direct contact with the mouth or nasal cavity, and has multiple cyclic transmission modes. It mainly exists in blood, tissue fluid, internal organs, secretions, and excretions. It has the characteristics of high infection rate, rapid spread, and high fatality rate. The pig industry has caused huge economic losses. [0003] The most important method o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/04C12N15/85C12N15/34A61K39/12A61P31/20C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K39/12A61P31/20C12N2710/12021C12N2710/12022C12N2710/12052C12N2710/12062C12N2710/12034C12N2800/107A61K2039/5254A61K2039/552
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
Owner JINYUBAOLING BIO PHARMA CO LTD
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