重组人早孕因子的表达与纯化  

Prokaryotic Expression and Purification of Early Pregnancy Factor Protein

作　　者：杜晓明[1] 张改平[1,2] 王丽[2] 郭军庆[2] 王林林[1] 冯华[1] 权凯[2] 王爱萍[1] 

机构地区：[1]郑州大学生物工程系,郑州450001 [2]河南省农业科学院农业部动物免疫学重点开放实验室河南省动物免疫学重点实验室,郑州450002

出　　处：《中国农学通报》2011年第14期40-44,共5页Chinese Agricultural Science Bulletin

基　　金：河南省科技攻关项目"早孕因子在奶牛超早期妊娠诊断的研究"(082300453208)

摘　　要：早孕因子(EPF)是具有免疫抑制和生长调节活性的妊娠相关蛋白,为目前最早确认妊娠的生化指标之一。为获得人早孕因子重组蛋白,通过PCR技术扩增人早孕因子基因,克隆入原核表达载体pGEX-6p-1,与GST基因相融合,构建重组表达质粒,转化大肠杆菌表达菌株BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,以SDS-PAGE和Western blot分析重组蛋白的表达,通过GST树脂纯化表达蛋白。结果成功构建重组表达质粒pGEX6P-EPF,在大肠杆菌中诱导表达了GST-EPF融合蛋白,表达蛋白分子量为37.3kDa,并能被抗GST单克隆抗体特异识别,GST亲和层析纯化,制备了EPF重组蛋白。Early pregnancy factor （EPF） is a pregnancy-associated protein that has immune-suppressive and growth-regulatory activities. It is regarded as a biochemical index by which super-early pregnancy can be determined. The objective of the study was to express the recombinant EPF protein in Escherichia coli （E. coli）. The EPF gene amplified by PCR was sub-cloned into a pGEX-6P-1 vector fused with GST gene to construct a recombinant plasmid. The recombinant plasmid was then transformed into E. coli BL21.The expression of recombinant protein was analyzed by SDS-PAGE and Western blot after induction by IPTG. The recombinant protein was purified by high-affinity GST resin chromatography. The prokaryotic expression plasmid pGEX6P-EPF for producing EPF protein was constructed and the recombinant protein with a molecular weight of 37.3 kDa was expressed successfully in E. coli, which was recognized by the anti-GST monoclonal antibody in Western blot analysis. The recombinant EPF protein was obtained after GST affinity purification.

关 键 词：早孕因子 原核表达 重组蛋白 

分 类 号：R714.1[医药卫生—妇产科学]