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

摘要/Abstract

摘要： 【目的】应用原核表达系统表达猪圆环病毒4型(Porcine circovirus type 4,PCV4)复制酶蛋白(Rep),纯化后免疫小鼠制备Rep蛋白多克隆抗体。【方法】以PCV4福建株(FJ2020001)为模板,采用PCR法扩增获得Rep基因片段,构建原核表达质粒pET-30a-Rep;将pET-30a-Rep转化大肠杆菌BL21(DE3)感受态细胞,用0.5 mmol/L IPTG诱导表达重组Rep蛋白,利用SDS-PAGE和Western blotting鉴定重组蛋白。表达产物经镍柱纯化后免疫BALB/c小鼠制备多克隆抗体,利用Western blotting和间接ELISA鉴定鼠源多克隆抗体的免疫原性和抗体效价。【结果】成功克隆PCV4 Rep基因,构建了可表达重组Rep蛋白的原核表达质粒;SDS-PAGE结果显示,表达的重组蛋白分子质量约为35 ku,主要以包涵体形式存在;Western blotting结果表明,该蛋白能被PCV4阳性血清和鼠源多克隆抗体特异性识别,制备的鼠源多克隆抗体效价可达1∶16 000。【结论】本研究成功表达并纯化了具备抗原性的PCV4重组Rep蛋白,为开发PCV4血清学诊断试剂盒和开展猪场PCV4流行病学调查奠定基础。

关键词: 猪圆环病毒4型(PCV4); Rep蛋白; 原核表达; 多克隆抗体

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

中图分类号:

S852.65⁺5

陈如敬, 吴学敏, 陈秋勇, 严山, 吴仁杰, 车勇良, 王隆柏, 周伦江. 猪圆环病毒4型Rep蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(10): 4188-4195.

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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