奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

本研究旨在表达、纯化奶牛早孕因子重组蛋白及制备多克隆抗体。本试验构建奶牛早孕因子重组蛋白原核表达载体pET32a-EPF,转化至大肠杆菌BL21(DE3)内进行诱导表达,SDS-PAGE切胶纯化的早孕因子重组蛋白免疫BALB/c小鼠制备多克隆抗体,并用Western blotting和ELISA对重组蛋白及抗体进行检测。结果显示,成功表达并纯化了奶牛早孕因子重组蛋白,重组蛋白分子质量约37ku,表达量约占菌体总蛋白的48.6%,纯化后目的蛋白纯度可达93%,重组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∶12 800。结果表明,本研究成功表达并纯化了奶牛早孕因子重组蛋白,为研究奶牛早孕诊断奠定基础。

Prokaryotic Expression,Purification of Early Pregnancy Factor Protein of the Dairy Cattle and Preparation of Polyclonal Antibody against it

The study was aimed to express and purify the dairy cattle early pregnancy factor (EPF) protein and prepare EPF polyclonal antibody. The recombinant plasmid pET32a-EPF was expressed in E.coli BL21 (DE3). Polyclonal antibodies were developed by immunizing BALB/c mice with the SDS-PAGE gel extraction purified fusion protein. The recombinant protein and the polyclonal antibody were separately detected by Western blotting and ELISA. The results showed that the recombinant protein with a molecular weight of 37 ku was expressed successfully in E.coli and its content was approximately 48.6% of total bacteria proteins. The purity of target protein could reach 93% after purification. The recombinant protein was recognized by the prepared polyclonal antibody in Western blotting analysis. The antibody titer was 1:12 800.These results indicated that we had successfully obtained and purified the dairy cattle EPF protein, which laid a foundation for the further study of dairy cattle pregnancy diagnosis.