Method for preparing African swine fever virus P30 and P54 yeast vaccine

A kind of African swine fever virus and virus technology, applied in the field of biogenetic engineering, can solve the problems of drug resistance of intestinal pathogens and achieve comprehensive and effective protection

Pending Publication Date: 2020-04-14
TIANJIN UNIV
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However, this receptor also transports IgG antibodies across mucosal surfac

Method used

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  • Method for preparing African swine fever virus P30 and P54 yeast vaccine
  • Method for preparing African swine fever virus P30 and P54 yeast vaccine
  • Method for preparing African swine fever virus P30 and P54 yeast vaccine

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Embodiment 1

[0031] 1. Materials and methods

[0032] 1.1 Materials

[0033] 1.1.1 Yeast strains: For yeast surface display, S. cerevisiae strain ST1814G was used.

[0034] In the present invention: ST1814G is based on the Saccharomyces cerevisiae strain JDY52 in the inventor's laboratory [11] Retrofit build and save.

[0035] The surface-displaying yeast strain used in the present invention is a host strain ST1814G constructed on the basis of Saccharomyces cerevisiae JDY52, which constitutively expresses the glucose promoter GPD and displays the Aga1 protein on the surface. The galactose promoter LAC of the PIU211 plasmid (Addgene product number 44036) was replaced by the glucose promoter GPD by conventional molecular manipulation, and the high-efficiency display Aga1 host strain ST1814G under the control of the constitutive glucose promoter GPD was constructed by yeast transformation, and the GPD promoter was reduced Reduced the induced expression cost of recombinant Saccharomyces cer...

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Abstract

The invention discloses a method for preparing an African swine fever virus P30 and P54 yeast vaccine. The vaccine contains African swine fever virus strong immunogen protein P30, membrane structure protein P54, Fc fragments of swine IgG1 and IgA1 (namely Fc gamma, Fc alpha), and His purification tags. The method comprises the following steps: S1, connecting ASFV immunogen protein genes p30 and p54 with Fc gamma and Fc alpha in series; S2, respectively constructing transcription units of p30-Fc gamma and p54-Fc alpha in fusion expression in the yeast; S3, realizing series connection of the twotranscription units through in-vitro enzyme linking, yeast in-vivo conversion and homologous recombination technologies, and stably integrating the two transcription units into surface display type saccharomyces cerevisiae; and S4, carrying out genome level and protein level verification, and confirming to obtain two recombinant yeast strains of which the independent surfaces show and express theP30-Fc gamma and P54-Fc alpha fusion proteins, and a tandem composite expression type recombinant yeast strain of which the surface shows and expresses the two fusion proteins at the same time in S1.The strain is mature in production technology, suitable for large-scale production, safe to use and capable of inducing remarkable mucous membrane and humoral immune response of target animals.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering and relates to a method for preparing African swine fever virus P30 and P54 yeast vaccines. Background technique [0002] African swine fever is a severe infectious disease of pigs caused by African swine fever virus (ASFV), which can cause a fatality rate of up to 100% in acute infection. Since it was first reported in Kenya in 1921 [1] , There have been outbreaks in dozens of countries and regions. my country first issued an announcement on the African swine fever epidemic in August 2018, causing billions of dollars in economic losses. It will also become a huge threat to my country's pig farming industry in the future. Due to its large genome (170-190k bp) and multiple genotypes, there are still many gaps in understanding and cognition of African swine fever virus, which seriously hinders the development of effective vaccines. [0003] Key structural proteins of ASFV, s...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N15/66C12N1/19A61K39/187A61P31/20C12R1/865
CPCC07K14/005C12N15/81C12N15/66A61K39/12A61P31/20C12N2710/12022C07K2319/30C12N2710/12034A61K2039/552
Inventor 黄金海张丽琳郭艳余陈琛
Owner TIANJIN UNIV
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