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Construction of E184L gene deletion attenuated African swine fever virus strain and application of E184L gene deletion attenuated African swine fever virus strain as vaccine

Active Publication Date: 2021-01-22
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, different genotypes use the same gene deletion strategy, and the attenuation effect is quite different; at the same time, incomplete deletion may cause immune side effects and field poisoning; deletion of some genes may cause the virus to fail to replicate; and excessive virulence caused by multiple gene deletions Weakness will cause the attenuated strain to lose its protective effect and other problems, which also limit the progress of the development of African swine fever vaccine

Method used

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  • Construction of E184L gene deletion attenuated African swine fever virus strain and application of E184L gene deletion attenuated African swine fever virus strain as vaccine
  • Construction of E184L gene deletion attenuated African swine fever virus strain and application of E184L gene deletion attenuated African swine fever virus strain as vaccine
  • Construction of E184L gene deletion attenuated African swine fever virus strain and application of E184L gene deletion attenuated African swine fever virus strain as vaccine

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Experimental program
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Effect test

Embodiment 1

[0077] Example 1 Detection of immunosuppressive activity of E184L protein

[0078] 1. Detection of E184L protein inhibiting poly(dA:dT)-induced IFN-β, ISRE and NF-κB promoter activity

[0079] Construction of E184L expression plasmid: the African swine fever virus E184L protein coding sequence was amplified by RT-PCR to construct the E184L expression plasmid. The forward primer is 5'-TTTTGCGGCCGCGATGTTGGTGATCTTCTTGGGAATT-3' (shown in SEQ ID NO: 3); the reverse primer is 5'-TTTCGTCGACTTAACTATTATTTTCTTTCCACTCT- 3' (shown in SEQ ID NO: 4); digested by NotI and SalI respectively The amplified fragment and the FLAG-CMV-7.1 vector were purified and recovered and ligated with T4 ligase, and the E184L coding sequence was constructed into the FLAG-CMV-7.1 vector to obtain the E184L expression plasmid. Plasmid DNA was extracted with an endotoxin-free plasmid extraction kit, the concentration was measured, and stored at -20°C for later use. According to gene sequence determination, the...

Embodiment 2

[0101] Example 2 Construction and purification identification of attenuated African swine fever virus strain with loss of function of E184L gene-encoded protein

[0102] Primary porcine alveolar macrophages (PAM) and primary bone marrow macrophages (BMDM) used in the present invention are obtained from 2-4 month old healthy pigs (purchased from the Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences), and the cells are aseptically collected Afterwards, red blood cells were removed with erythrocyte lysate (purchased from Biosharp), after low-speed centrifugation, the supernatant was discarded, and the cells were resuspended in RPMI 1640 complete medium (purchased from Gibco) containing 10% FBS (purchased from PAN) in 37°C, 5% CO 2 cultured in an incubator. BMDM cell culture requires an additional 10ng / mL final concentration of recombinant porcine GM-CSF (purchased from R&D Systems) in RPMI 1640 complete medium, placed at 37°C, 5% CO...

Embodiment 3

[0114] Example 3 Virus titer determination of attenuated African swine fever virus strain ASFVΔE184L in PAM cells

[0115] The titration of African swine fever virus adopts half hematosorbate amount (50% haemadsorption, HAD 50 ) in two ways. Dilute the above-mentioned recombinant virus by 10 times, take 100 μl and inoculate the porcine primary PAM cells in 96 wells that have been laid in advance to form a single layer, add 25 μl of 1% red blood cell suspension to each well, the virus infection can be based on the accumulation of red blood cells around the infected cells The formed rosettes are judged, and the plate can be read for the first time after 12-16 hours of culture, and the observation is continued for 7 days, and the half hemasorbed dose (HAD) is calculated according to the Reed and Muench method. 50 ). The results showed that: 48 hours after infection, the average titer of the attenuated African swine fever virus strain ASFVΔE184L was 10 4.5 / mL, while the averag...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to construction of an E184L gene deletion attenuated African swine fever virus strain and an application of the E184Lgene deletion attenuated African swine fever virus strain as a vaccine. The inventor firstly finds that an African swine fever virus E184L protein can inhibit the activation of poly (dA: dT)-induced IFN-beta, NF-kappa B and ISRE promoters, inhibits generation of poly (dA: dT)-induced IFN-beta and downstream factors ISG15, ISG54, IL-6, MCP1, TNF-alpha and other cytokines, has strong immunosuppressive effect, and can be used as an immunosuppressive; secondly, the immunosuppressive function of an E184L gene encoding protein is deleted from the African swine fever parent strain ASFV CN / GS / 2018 through a genetic engineering means, so that the immunosuppressive activity of the parent strain is reduced, and an attenuated African swine fever candidate vaccine strain with high safety is obtained; and after the attenuated African swine fever candidate vaccine strain is used for immunizing pigs, the immunosuppressive activity and pathogenicity of the pigs are reduced, remarkable neutralizing antibodies can be induced to be generated after immunization, and the attenuated African swine fever candidate vaccine strain can be used as a safe and effective candidate vaccine strain for preventing and controlling African swine fever epidemic situations and has great social value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of an attenuated African swine fever virus strain with E184L gene deletion and its application as a vaccine. Background technique [0002] African swine fever (African swine fever, ASF) is a contagious, extensive hemorrhagic and severe infectious disease of pigs caused by African swine fever virus (ASFV). The sick pigs have elevated body temperature and difficulty breathing , extensive mucosal bleeding and other clinical symptoms, with a short course of disease and high fatality rate. The virulent strain can cause domestic pigs to die within 5-14 days after infection, and the mortality rate is close to 100%. It is the most serious threat to the healthy development of the pig industry in the world. one of the infectious diseases. The ASF epidemic is spreading globally, and the epidemic in neighboring countries is increasing year by year, and there are nat...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K39/12A61P37/06A61P31/20C12N7/04C12N15/85
CPCA61K38/162A61K39/12A61P37/06A61P31/20C12N7/00C12N15/85C07K14/005A61K2039/552A61K2039/5254A61K2039/54A61K2039/545C12N2710/12021C12N2710/12022C12N2710/12034Y02A50/30
Inventor 郑海学朱紫祥李莎莎党文杨帆曹伟军杨金平刘会胜刘华南田宏张克山孙研刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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