猪细环病毒2型衣壳蛋白的原核表达及纯化

本试验通过在大肠杆菌中表达衣壳蛋白，确定最优表达条件并对其进行纯化。用PCR法扩增猪细环病毒2型（TTV2）ORF1抗原基因，定向克隆至质粒pGEX-4T-1中，获得重组质粒pGEX-4T-1-ORF1。经双酶切鉴定及测序正确后转化到BL21(DE3)工程菌中，获得衣壳蛋白大肠杆菌表达株。IPTG诱导表达，表达的重组融合蛋白经SDS-PAGE及Western blotting鉴定，并对影响重组蛋白表达的3个因素，即诱导时间、诱导温度和IPTG浓度进行优化，确定最优表达条件。重组表达质粒PCR及双酶切鉴定结果显示衣壳蛋白原核表达载体构建正确。重组融合蛋白分子质量约80 ku，主要以包涵体形式存在。37 ℃诱导5 h表达量最多，IPTG浓度对表达量无明显影响。蛋白质串联肽谱分析检测氨基酸序列与GenBank中公布的TTV2 ORF1基因序列翻译的氨基酸序列相对应，且可被鼠抗GST单克隆抗体特异性识别。该融合蛋白首次用KCl染色切胶回收法对表达的融合蛋白进行纯化，纯化效果较好，性价比高。结果表明，成功构建了pGEX-4T-1-ORF1重组质粒，衣壳蛋白得到了高效表达及纯化，为间接ELISA的建立及单克隆抗体的研制提供了抗原。

Prokaryotic Expression and Purification of the Cap Protein of Porcine Toque Teno Virus Type 2

The study was aimed to highly express Cap protein of porcine torque teno virus type 2 in E.coli, and determine the optimal expression conditions and purify it. ORF1 gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4T-1 to construct recombinant plasmid pGEX-4T-1-ORF1, which was identified by restriction and sequence analysis, then transformed into E.coli BL21(DE3) and expressed under the induction of IPTG. The expression product was identified by SDS-PAGE and Western blotting. The induction time, induction temperature and IPTG concentrations were optimized. Both PCR and restriction analysis proved that the recombinant plasmid was constructed correctly. The best induction condition was 37 ℃ for 5 h, whereas IPTG concentration had no significant impact on protein expression. The recombinant fusion protein pGEX-4T-1-ORF1 was approximately 80 ku in a form of inclusion body, which was recognized specially by mouse anti-GST monoclonal antibody. It was the first time to use cutting the gel slice’s method for purification of TTV2 Cap protein, which was cost-effective and convenient. Recombinant plasmid pGEX-4T-1-ORF1 was constructed and the Cap protein was highly expressed in E.coli which provided antigen for indirect ELISA and preparation of monoclonal antibodies.