非洲猪瘟病毒B438L蛋白的原核表达及其多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2021.03.023

Abstract

Abstract: The purpose of this study was to express the recombinant protein p49 via constructing the prokaryotic expression system of African swine fever virus (ASFV) B438L gene,then prepare the polyclonal antibody against p49 protein.B438L gene was synthesized according the gene sequence of ASFV Georgia 2007/1 (GenBank accession No.:FR682468.1),then inserted into pET-28a(+) vector to construct pET-28a-B438L recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,then induced by 1 mmol/L IPTG at 16 ℃ for 12 h,the recombinant protein expression form was identified by SDS-PAGE.MALDI-TOF/TOF was used to determine whether the recombinant protein was expressed correctly.The polyclonal antibody against p49 was prepared by immunizing mice with the purified recombinant protein p49,the antibody titer was detected by indirect ELISA,and the specificity was identified by indirect immunofluorescence assay (IFA) and Western blotting.The results showed the pET-28a-B438L was successfully constructed.Then the recombinant plasmid was transformed into E.coli BL21(DE3),and the recombinant protein was expressed mainly in the form of inclusion bodies,with mass at about 60 ku.The recombinant protein was further confirmed by tandem mass spectrometry.The indirect ELISA result showed that the titer of polyclonal antibodies was as high as 1:64 000.The results of IFA and Western blotting indicated that the polyclonal antibody could specifically recognize the recombinant protein p49.These results confirmed the recombinant protein p49 expressed via prokaryotic system had good immunogenicity,and the prepared polyclonal antibodies had high titer and specificity.This study provided technical support for further study of the biological structure and function of ASFV p49 protein for the further ASFV related diagnosis and vaccine development.

Key words: African swine fever virus (ASFV); B438L gene; p49 recombinant protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺1

LIU Xueting, WANG Zhaoyang, XIN Ting, GUO Xiaoyu, JIANG Yitong, CUI Shuai, YU Hainan, ZHU Hongfei, JIA Hong. Prokaryotic Expression and Polyclonal Antibody Preparation and Identification of African Swine Fever Virus B438L Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 991-1000.

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Prokaryotic Expression and Polyclonal Antibody Preparation and Identification of African Swine Fever Virus B438L Protein

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