猪圆环病毒3型Cap蛋白的原核表达及其免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of Porcine Circovirus Type 3-Cap Protein

为了表达猪圆环病毒3型(PCV3)衣壳(Cap)蛋白,分析其免疫原性,本试验构建了不包含Cap蛋白核定位序列的原核表达质粒pET-28a-PCV3-Cap,应用大肠杆菌Escherichia coli表达系统诱导表达了重组Cap蛋白;通过SDS-PAGE、Western Blot及液相色谱-质谱联用(LC-MS/MS)技术对表达蛋白进行了鉴定;分析预测了蛋白的抗原表位,并通过动物试验分析蛋白的免疫原性。结果显示,在31℃下经0.5 mmol/L的IPTG诱导4 h,蛋白(25.4 kDa)表达量最高,其主要以包涵体形式表达;表达蛋白与PCV3参考毒株Cap蛋白氨基酸序列的一致性为88.4%(152/172 aa),并且预测的抗原表位与参考毒株的相符;重组蛋白能够刺激小鼠产生特异性抗体,平均抗体水平≥1∶12800。上述结果表明,表达的蛋白具有良好的免疫原性。该试验为深入解析PCV3-Cap蛋白抗原表位的免疫活性以及相关亚单位疫苗和特异性抗体检测技术的研究提供了科学依据。

In order to express porcine circovirus type 3(PCV3)capsid(Cap)protein and analyze its immunogenicity,recombinant expression plasmids(pET-28a-PCV3-Cap)without the nuclear localization region of Cap protein were constructed and Escherichia coli expression system was employed for express the recombinant protein.The recombinant protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),Western Blot and liquid chromatography-tandem mass spectrometry(LC-MS/MS).The antigenic epitopes of the recombinant Cap protein were prediced and the immunogenicity of the protein was evaluated by the animal experiment.The protein(25.4 kDa)mainly in the form of inclusion bodies was expressed in the highest level after induction with 0.5 mmol/L isopropyl-β-d-thiogalactoside(IPTG)for 4 h at 31℃.The identity was 88.4%(152/172 aa)between the amino acid sequence of the expressed protein and that of the reference sequence of PCV3-Cap protein.Predicted antigenic epitopes on the recombinant protein corresponded with those on the reference sequence.The recombinant protein stimulated mice to produce PCV3-Cap specific antibodies with an average titer≥1:12800.The results showed that the recombinant Cap protein in the study owns favorable immunogenicity,which will lay a solid foundation for further analysis of immune activities of the antigenic epitopes on the PCV3-Cap protein as well as correlational research on subunit vaccine and specific detection techniques.