细粒棘球六钩蚴副肌球蛋白的原核表达、纯化及多克隆抗体的制备

摘要

为了表达、纯化细粒棘球六钩蚴副肌球蛋白及制备多克隆抗体,本研究构建细粒棘球六钩蚴副肌球蛋白原核表达载体pET30a-M26,转化至大肠杆菌BL21 (DE3)内进行诱导表达,SDS-PAGE切胶纯化的副肌球蛋白,免疫Balb/c小鼠制备多克隆抗体,并用Western blotting和ELISA对副肌球蛋白及抗体进行检测.结果显示:成功表达并纯化了细粒棘球六钩蚴副肌球蛋白,重组蛋白分子质量约41 kD,纯化后目的蛋白纯度可达85％,蛋白浓度为0.5mg/ml.副肌球蛋白可与所制备的多克隆抗体结合,ELISA测定的抗体效价为1∶12800,结果表明,成功表达并纯化了六钩蚴副肌球蛋白.本研究可为研究包虫病早期诊断奠定了基础.%In order to express and purify the paramyosin of oncosphere in Echinococcus granulosus,and to prepare its polyclonal antibody,the prokaryotic expression vector pET30a-M26 for the paramyosin was constructed,which was transformed into Escherichia coli (E.coli) BL21 (DE3) and induced expression of the paramyosin.The protein was purified by SDS-PAGE and then immunized with Balb/c mice to prepare polyclonal antibody.Western blotting and ELISA were used to detect paramyosin and antibody,respectively.The results showed that the recombinant protein was about 41 kD,and the purity of the protein was up to 85％ and the protein concentration was 0.5 mg/mL.The paramyosin could bind to the prepared polyclonal antibody and ELISA detected the titer of the antibody was 1:12800.The results suggest that the paramyosin can be successfully expressed and purified.This study can lay the foundation for the early diagnosis of hydatid disease.