粗糙脉孢菌ERG-11蛋白抗原的原核表达、纯化及其多克隆抗体制备

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Neurospora crassa ERG-11 Protein Antigen

构建p ET-28a(+)-ERG-11重组质粒,表达6×His-ERG-11融合蛋白,制备ERG-11多克隆抗体。采用PCR技术扩增目的片段,插入p ET-28a(+)原核表达载体,并转入E.coli BL21(DE3)感受态表达融合蛋白,融合蛋白经亲和纯化及分子筛纯化后免疫新西兰大白兔制备多克隆抗体,取血清后,采用间接ELISA法和Western blot法检测多克隆抗体的效价及特异性。成功构建了p ET-28a(+)-ERG-11表达载体,SDS-PAGE电泳显示成功诱导出以包涵体形式存在的6×His-ERG-11融合蛋白,两步纯化后得到纯度较高的抗原,间接ELISA法显示制备的多克隆抗体效价达到1∶512 000,Western blot显示具有较高特异性。成功实现了粗超脉孢菌ERG-11蛋白的原核表达,制备出一支兔抗粗超脉孢菌ERG-11的多克隆抗体。

This work aims to construct the recombinant plasmid p ET-28a（＋）-ERG-11 and express the 6 × His-ERG-11 fusion proteinfor preparing ERG-11 polyclonal antibody. The target fragment amplified by PCR was inserted into p ET-28a（＋）prokaryotic expressionvector,and then transformed into the competent cells of Escherichia coli BL21（DE3）for expressing the fusion protein. Further,the fusionprotein was purified by affinity purification and gel filtration chromatography,and then immunized to New Zealand white rabbit for preparingpolyclonal antibody. Taking serum,the titer and specificity of the polyclonal antibody were detected through indirect ELISA and Westernblot,respectively. As results,the p ET-28a（＋）-ERG-11 expression vector was successfully constructed. SDS-PAGE showed that the 6× His-ERG-11 fusion protein was induced successfully in the inclusion form. After two-step purification,the antigen with high purity wasobtained. ELISA showed that the polyclonal antibody titer reached 1∶512 000,and Western blot confirmed its high specificity. Conclusively,the prokaryotic expression of ERG-11 gene was successfully conducted,and a polyclonal antibody against Neurospora crassa ERG-11 wasprepared,providing a foundation for further study on the genetic structure and function of ERG-11 in N. crassa.