非洲猪瘟病毒主要抗原p54-1的原核表达及多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2018.10.019

Abstract

Abstract:

In order to further study the major epitopes and antigen properties of African swine fever virus (ASFV) p54-1 protein,specific primers were designed according to p54 gene sequence retrieved from GenBank (accession No.:NC_001659.2),the target sequence of encoding p54-1 protein was amplified by PCR.Then it was ligated into pGEX6p-1 vector and constructed prokaryotic expression plasmid (pGEX6p-1-p54-1).The plasmid was transfected into E.coli BL21,and the expression of recombinant protein was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The fusion protein cleaved the GST-tag using thrombin,and then purified by anion exchange column.The protein was identified by ELISA and emulsify with adjuvant,the prepared immunogen was inoculated into mouse to prepare of p54-1 protein specific polyclonal antibody.The immune-activity and titers of the prepared polyclonal antibody were determined by ELISA and Western blotting.The results showed that the expressed recombinant protein p54-1 existed in a soluble form.The p54-1 protein cleaved GST-tag could react with the positive serum of ASFV,but no with negative serum,PRRSV and PCV3.The prepared polyclonal antibody titer was 1:128 000.Western blotting result demonstrated that the prepared polyclonal antibody could recognize the p54 protein.In conclusion,the high purified expressed protein of ASFV p54-1 had been successfully prepared and p54-1 specific polyclonal antibody showed wonderful immunocompetence and specificity,providing foundation for the development of sandwich ELISA detection method of ASF.

Key words: African swine fever virus (ASFV); p54 protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺1

WANG Caixia, FENG Chunyan, DU Fangyuan, LIU Dandan, ZHANG Yongning, LIN Xiangmei, WU Shaoqiang. Prokaryotic Expression of African Swine Fever Virus p54-1 and Preparation and Characterization of Its Polyclonal Antibody[J]. , 2018, 45(10): 2823-2830.

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Prokaryotic Expression of African Swine Fever Virus p54-1 and Preparation and Characterization of Its Polyclonal Antibody

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