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Construction of African swine fever attenuated strain with polygene joint deletion and application of African swine fever attenuated strain with polygene joint deletion, as vaccine

An African swine fever virus and gene deletion technology, applied in the construction and application fields of vaccines, can solve the problems of insufficient pathogenicity, reduced immunogenicity or protective effect of attenuated strains, residues, etc.

Active Publication Date: 2021-08-06
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, the virulence gene knockout vaccine of classical swine fever virus also considers the following factors: (1) Whether the knockout virulence gene reduces virus transmission, detoxification, causes tissue damage and affects production performance is a factor of biosafety. An important indicator, which is also an important indicator for the continuous improvement of the safety of gene deletion vaccines; (2) Different strains have different effects of deleting the same gene, and insufficient deletion has residual virulence and pathogenicity problems; and viruses caused by multiple gene deletions Low titer will also reduce the risk of immunogenicity or protective effect of the attenuated strain; (3) Although the whole genome sequencing of African swine fever has been completed, there are more than 160 regulatory genes and structural genes that make up the African swine fever virus Although the project is huge, it is crucial to the function of regulatory genes and structural genes for its pathogenic mechanism and vaccine development

Method used

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  • Construction of African swine fever attenuated strain with polygene joint deletion and application of African swine fever attenuated strain with polygene joint deletion, as vaccine
  • Construction of African swine fever attenuated strain with polygene joint deletion and application of African swine fever attenuated strain with polygene joint deletion, as vaccine
  • Construction of African swine fever attenuated strain with polygene joint deletion and application of African swine fever attenuated strain with polygene joint deletion, as vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction and purification identification of recombinant virus Δ20 / 53

[0057] 1. Screening expression cassette construction

[0058] To facilitate screening, an expression cassette for the selectable marker gene was constructed.

[0059]Enhanced green fluorescent protein (Enhance Green fluorescent protein, eGFP) gene screening expression cassette construction: reference (O'Donnell V, Holinka LG, Krug PW, Gladue DP, Carlson J, Sanford B, Alfano M, Kramer E, Lu Z, Arzt J, Reese B, Carrillo C, Risatti GR, Borca MV. African Swine Fever Virus Georgia 2007with a Deletion of Virulence-Associated Gene9GL(B119L), when A administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Prevention HomologousChallenge.JVirol.2015; 89(16):8556-66), the p72 promoter was amplified by PCR (from -196nt upstream of the p72 gene to the sequence before +17), spare; the amplification primer was: forward primer 5' -TTATAA AACATATGTTCATAAAAAGGGTCGCCGGAGGA...

Embodiment 2

[0072] The mensuration of embodiment 2 virus titers

[0073] The titer of African swine fever virus adopts half hematosorbate amount (50% haemadsorption, HAD 50 ) means that HAD 50For the specific operation of the experiment, please refer to the literature (Borca MV, Ramirez-Medina E, Silva E, Vuono E, Rai A, PruittS, Holinka LG, Velazquez-Salinas L, Zhu J, GladueDP. Development of a highly effective African an swine fever virus vaccine by deletion of the I177L generesults in sterile immunity against the current epidemic Eurasiastrain.JVirol.2020.pii:JVI.02017-19), and adjust it appropriately: in 96-well plate according to about 1x10 5 Cells per well were inoculated with primary PAM cells, and the recombinant virus to be tested was serially diluted 10 times, with a total of 7 dilutions, each dilution had 8 wells, and the diluted virus was added to the PAM of a 96-well plate at 100 μL / well, and then added Red blood cells, a total of three replicates. Virus infection can be j...

Embodiment 3

[0074] Toxicity Evaluation of Example 3 Gene Deletion Strain Δ20 / 53

[0075] In order to detect the virulence of the gene deletion strain Δ20 / 53, 10 4 HAD 50 Dose was injected intramuscularly to evaluate its toxicity in piglets.

[0076] In this experiment, 12 African swine fever antigen-negative healthy Landrace piglets were divided into 2 groups, including 6 pigs in the gene deletion strain Δ20 / 53 immunization group and 6 pigs in the cohabitation group. Body temperature was measured every day after immunization, and peripheral blood and saliva were collected, referring to literature (King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, Bastos A D, Drew TW.2003. Development of a TaqMan PCR assay with internal amplification control ol for the detection of African swine fever virus. J VirolMethods 107:53-61), and the ASFV virus content in the blood was measured by fluorescent quantitative PCR method, and the detection was terminated after 17 days. The lethali...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an attenuated African swine fever virus with immunosuppressive gene deletion, and a vaccine. According to the invention, an ASFV MGF-110-9L gene and an ASFV MGF-505-7R gene are found to have a relatively strong immunosuppression effect, the virulence of the African swine fever virus can be weakened by knocking out the genes from the African swine fever virus, and the genes are new target genes for preparing attenuated swine fever vaccines. The functions of ASFV G-ACD-00190 gene coding proteins, ASFV MGF-110-9L gene coding proteins, ASFV G-ACD-00210 gene coding proteins and ASFV MGF-505-7R gene coding proteins are deleted in an African swine fever parent strain ASFV CN / GS / 2018 in a combined manner by adopting a genetic engineering means, so that the toxicity of the parent strain is reduced, and the attenuated African swine fever candidate vaccine strain with high safety is obtained. The attenuated African swine fever candidate vaccine strain is completely attenuated after immunizing a pig, the immunized pig is healthy, 100% immune protection can be provided for attacking (100HAD50 dosage) of ASFV CN / GS / 2018 virulent strain, and the attenuated African swine fever candidate vaccine strain can be used as a safe and effective candidate vaccine for preventing and controlling the African swine fever epidemic situation and has great social value.

Description

[0001] This application claims the priority of the previous application with the application date of July 10, 2020, the application number CN 2020106621078, and the invention titled "Construction of African swine fever multi-gene combined deletion attenuated strain and its application as a vaccine". The entire content of the earlier application is incorporated in the present application. technical field [0002] The invention belongs to the technical field of bioengineering, and in particular relates to the construction of an African swine fever multi-gene combined deletion attenuated strain and its application as a vaccine. Background technique [0003] African swine fever (African swine fever, ASF) is caused by the infection of African swine fever virus (ASFV), and is characterized by fever and hemorrhage in pigs. The fatality rate for domestic pigs is as high as 100%. . [0004] At present, there are three main methods for the preparation of African swine fever vaccine: ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/34A61K39/187A61P31/20
CPCC12N7/00C07K14/005A61K39/12A61P31/20C12N2710/12021C12N2710/12022C12N2710/12034A61K2039/5254A61K2039/552
Inventor 郑海学李丹党文张克山刘华南申超超冯涛李攀齐晓兰张敬刘迎琦田宏茹毅杨帆曹伟军何继军刘志杰张伟郭建宏殷宏孙研刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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