非洲猪瘟病毒360-12L蛋白的原核表达及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.01.037

Abstract

Abstract: [Objective] The study was aimed to obtain the antigenic 12L protein of African swine fever virus (ASFV) used to serological diagnosis of ASFV. [Method] The 360-12L gene was synthesized and inserted into pET-28a(+) vector to construct prokaryotic expression plasmid pET-28a-12L. The correct plasmid identified by sequencing was transferred into E. coli BL21(DE3) competent cells for induction. The optimal induction temperature was selected and its solubility was analyzed. The recombinant protein 12L was purified by affinity chromatography and analyzed by SDS-PAGE. The recombinant protein 12L was used as immunogen to immunize female BALB/c mice to obtain mouse polyclonal antibody against ASFV 12L protein. The titer of the polyclonal antibody was determined by indirect ELISA, and its specificity was verified by indirect immunofluorescence assay. [Result] The prokaryotic expression plasmid of ASFV 12L was successfully constructed, and the recombinant strain pET-28a-12L-BL21 had a high expression level at 37 ℃ for 6 h, mainly in the form of inclusion body, with an obvious band at 54.7 ku, which was consistent with the expectation. Indirect ELISA showed that the titer of the polyclonal antibody against ASFV 12L protein was higher than 1:512 000. Indirect immunofluorescence assay showed that the polyclonal antibody against ASFV 12L protein had good specificity and could specifically recognize recombinant protein 12L. [Conclusion] The recombinant protein 12L expressed in E. coli had good immunogenicity, and the polyclonal antibody against ASFV 12L protein prepared from mice had high reactivity and specificity, which could lay a foundation and provide biomaterials for further study on biological function of ASFV recombinant protein 12L, serological diagnosis technology of ASFV and vaccine research.

Key words: African swine fever virus (ASFV); prokaryotic expression; recombinant protein 12L; polyclonal antibody

CLC Number:

S852.65⁺1

JIANG Yajun, CHEN Shiyu, XIN Ting, GUO Xiaoyu, WANG Zhaoyang, LIU Xueting, CUI Shuai, WANG Yang, ZHU Hongfei, JIA Hong. Prokaryotic Expression of African Swine Fever Virus 360-12L Protein and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 352-360.

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Prokaryotic Expression of African Swine Fever Virus 360-12L Protein and Preparation of Its Polyclonal Antibody

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