猪圆环病毒4型Rep蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.10.033

Abstract

Abstract: 【Objective】 The objective of the experiment was to express the replicase protein (Rep) of Porcine circovirus type 4 (PCV4) by prokaryotic expression system,and immunize mice with purified Rep protein to prepare polyclonal antibody.【Method】 The PCV4 Fujian strain (FJ2020001) was used as the template,the Rep gene fragment was amplified by PCR, and the prokaryotic expression plasmid pET-30a-Rep was constructed.pET-30a-Rep was transformed into Escherichia coli BL21 (DE3) competent cells,and 0.5 mmol/L IPTG induced the expression of recombinant Rep protein.SDS-PAGE and Western blotting were used to identify recombinant proteins.The expression products were purified by nickel column and immunized BALB/c mice to prepare polyclonal antibodies.Western blotting and indirect ELISA were used to identify the immunogenicity and antibody titer of the murine polyclonal antibodies.【Result】 PCV4 Rep gene was cloned successfully,and the prokaryotic expression plasmid was constructed which could express recombinant Rep protein.SDS-PAGE results showed that the molecular weight of the recombinant protein was about 35 ku,mainly in the form of inclusion.Western blotting results showed that the protein could be specifically recognized by PCV4 positive serum and murine polyclonal antibody,and the prepared murine polyclonal antibody titer could reach 1∶16 000.【Conclusion】 The recombinant Rep protein of PCV4 with antigenicity was successfully expressed and purified,which could lay a foundation for the development of PCV4 serological diagnosis kit and PCV4 epidemiological investigation in pig farms.

Key words: Porcine circovirus type 4 (PCV4); Rep protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺5

CHEN Rujing, WU Xuemin, CHEN Qiuyong, YAN Shan, WU Renjie, CHE Yongliang, WANG Longbai, ZHOU Lunjiang. Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Circovirus Type 4 Rep Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4188-4195.

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Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Circovirus Type 4 Rep Protein

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