鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.06.024

Abstract

Abstract: 【Objective】 The purpose of this study was to express the Capsid protein of Duck Tembusu virus (DTMUV) by prokaryotic expression system and prepare its polyclonal antibody to lay a foundation for the study of the molecular mechanism of DTMUV.【Method】 According to the gene sequence of DTMUV-201909 strain, the Capsid gene was cloned into the prokaryotic expression vector pET-30a(+) by one-step cloning technique.The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells.The recombinant protein was induced by IPTG.The recombinant protein was identified by SDS-PAGE and Western blotting.The purified recombinant protein was emulsified with ISA206 adjuvant at equal volume and immunized BALB/c mice to obtain polyclonal antibodies.The titer of the polyclonal antibody obtained was measured by indirect ELISA, and the specificity of the polyclonal antibody was identified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The recombinant plasmid pET-30a-Capsid was successfully obtained, SDS-PAGE showed that the molecular weight of the expressed recombinant protein was about 18 ku, mainly in the form of inclusion body.Western blotting results showed that the protein could react specifically with anti-His labeled mouse monoclonal antibody and had good reactivity.The results of indirect ELISA showed that the titer of the prepared polyclonal antibody against Capisd protein reached 1:256 000.Western blotting and IFA results showed that the polyclonal antibody could react specifically with DTMUV.【Conclusion】 The mouse anti-Capsid protein polyclonal antibody was successfully prepared.It provided materials for studying the structure and function of Capsid protein, and laid a foundation for revealing the pathogenesis of DTMUV.

Key words: Duck Tembusu virus (DTMUV); Capsid protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65

JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.

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Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein

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