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Application of African swine fever virus D205R and D345L genes

An African swine fever virus, D205R technology, applied in the field of biomedicine, can solve problems such as missing vaccine development

Active Publication Date: 2021-08-27
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This characteristic of multi-gene family makes most of the current research focus on the impact of deletion of multiple multi-gene family members on virus virulence and the development of vaccines

Method used

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  • Application of African swine fever virus D205R and D345L genes
  • Application of African swine fever virus D205R and D345L genes
  • Application of African swine fever virus D205R and D345L genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. African swine fever virus D205R and D345L genes can significantly inhibit virus infection-induced interferon promoter activity and interferon-stimulated response element activity

[0023] The invention uses the nucleotide sequences of D205R and D345L genes of African swine fever virus China / 2018 / AnhuiXCGQ (GenBank accession number: MK128995.1) as a template, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and successfully constructed on the pCAGGS vector , named pCAGGS-D205R and pCAGGS-D345L respectively. After the plasmids were constructed, they were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. After the sequencing was correct, subsequent cell experiments were carried out.

[0024]293T cells were revived and cultured in DMEM (Gibco) medium containing double antibodies (100 units / mL penicillin and 100 units / mL streptomycin) and 10% fetal bovine serum (Gibco). The 293T cells were spread evenly into 24-well plates and cultured in a ce...

Embodiment 2

[0025] Embodiment two, African swine fever virus D205R and D345L genes can inhibit the transcription of IFN-β

[0026] First spread 293T cells into 12-well plates, and when the cells grow to a confluence of 80-90%, transfect 293T cells transiently with 4 μg of pCAGGS-D205R and pCAGGS-D345L respectively according to VigoFect instructions, and change after 4-6 hours into a complete culture medium. After 24 hours of transfection, the cells were stimulated with SeV (100HAU / ml) for 12 hours, and then the cells were lysed with TRIzol (brand: MNG, product number: 740404.200), and RNA was extracted according to the instructions, and used as templates for fluorescent quantitative PCR (qPCR) after reverse transcription . According to qPCR (Beijing Quanshijin Biotechnology Co., Ltd., The recommended system of Green qPCRSuperMix) manual was used for fluorescence quantitative PCR experiment. The experimental results were analyzed with GraphPad Prism8. The results show that if figure...

Embodiment 3

[0027] Embodiment three, African swine fever virus D205R and D345L gene can suppress the expression of interferon-stimulated gene ISGs

[0028] First spread 293T cells into 12-well plates, and when the cells grow to a confluence of 80-90%, transfect 293T cells transiently with 4 μg of pCAGGS-D205R and pCAGGS-D345L respectively according to VigoFect instructions, and change after 4-6 hours into a complete culture medium. After 24 hours of transfection, the cells were stimulated with SeV (100HAU / ml) for 12 hours, and then the cells were lysed with TRIzol, and RNA was extracted according to the instructions, and used as templates for fluorescent quantitative PCR (qPCR) after reverse transcription. Fluorescent quantitative PCR experiments were performed according to the recommended system in the qPCR instructions. The experimental results were analyzed with GraphPad Prism8. The results show that if image 3 As shown, both D205R and D345L proteins can inhibit the transcription o...

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Abstract

The invention discloses application of African swine fever virus D205R and D345L genes, and particularly relates to application of the African swine fever virus D205R and D345L genes in preparation of an African swine fever virus vaccine. The African swine fever virus D205R and D345L genes can significantly inhibit virus infection induced interferon expression, and inhibit interferon stimulation gene ISGs expression. Through dual-luciferase reporter gene system detection, the invention finds that through comparison, sendai-virus-induced interferon promoter activity and interferon stimulation response element (ISRE) activity can be inhibited through expression of the D205R and D345L genes. The fluorescent quantitative PCR test finds that the expression of the D205R and D345L genes can significantly inhibit the mRNA expression level of I-type interferon IFNbeta induced by Sendai virus infection, and inhibit the expression level of interferon stimulating genes ISG15 and OASL playing an antiviral function; and in conclusion, two kinds of African swine fever virus proteins with the function of inhibiting host cell antiviral innate immune response are identified, and a new choice is provided for preparing an African swine fever virus gene deletion vaccine.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the functional identification of two viral proteins and their application in the preparation of gene deletion vaccines, and mainly relates to the functional identification of African swine fever virus D205R and D345L proteins and their application in the preparation of African swine fever virus gene deletion vaccines in the application. Background technique [0002] African swine fever virus (ASFV) is a DNA virus and the only member of the African swine fever virus family, the genus ASFV. African swine fever (African swine fever, ASF) is a severe infectious disease of pigs caused by ASFV, which can cause 100% death of infected domestic pigs. ASF first broke out in Kenya in the 1920s, then spread rapidly in South and East Africa, and was introduced to the Indian Ocean region in 1998. After the first outbreak outside Africa (Portugal) in 1957, ASF outbreaks were subsequently reported in ...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/20
CPCA61K39/12A61P31/20C12N2710/12034A61K2039/53Y02A50/30
Inventor 陈吉龙池晓娟
Owner FUJIAN AGRI & FORESTRY UNIV
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