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

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

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。

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.