【摘要】 为了获取大量具有免疫原性的早孕因子(EPF)重组蛋白,通过PCR技术扩增得到Hela细胞EPF基因片段,与pREST-A连接。将质粒pREST-A-EPF转化大肠埃希菌BL21后IPTG进行诱导表达,采用His标签蛋白纯化试剂盒纯化重组蛋白后,再用SDS-PAGE和Western blot分别鉴定其纯度和特异性。结果表明,EPF cDNA以正确的阅读框架插入表达载体pREST-A,经IPTG诱导后高效表达EPF重组蛋白,Western blot表明其与抗EPF抗体特异性结合。论文构建了EPF的原核表达载体,纯化获得了重组蛋白,为进一步开展EPF蛋白的相关研究和猪早孕诊断试剂盒的开发奠定了基础。更多还原

【Abstract】 In order to obtain a large amount of the recombinant protein with good immunogenicity of early pregnancy factor(EPF),EPF gene segment was firstly amplified by PCR.The objective gene fragment and the pREST-A vector were digested,connected and transformed into competent cells of Escherichia coli DH5α.By inducing the pregnancy plasmid with IPTG and culturing the transformed cells,the objective protein was expressed and then was purified using the His tagged protein purification kit.The purity of recombinant protein was detected by SDS-PAGE and its specificity was detected by Western blot;respectively.The results showed that EPF cDNA with the correct reading frame was inserted into expression vector PREST-A and high effective expression was obtained after inducing with IPTG.Western blot showed that the specific binding with anti EPF antibody was found.In this experiment we successfully constructed prokaryotic expression vector for EPF,purified and obtained the recombinant protein with special biological activity.The tesults contributes a foundation for further study of EPF protein.更多还原