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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (14): 40-44.

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

重组人早孕因子的表达与纯化

杜晓明   

  • 收稿日期:2011-01-12 修回日期:2011-02-09 出版日期:2011-06-15 发布日期:2011-06-15
  • 基金资助:

    河南省科技攻关项目

Prokaryotic Expression and Purification of Early Pregnancy Factor Protein

  • Received:2011-01-12 Revised:2011-02-09 Online:2011-06-15 Published:2011-06-15

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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，利用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，利用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。

关键词: 水稻, 水稻, 氮离子注入, 诱变效应, 抗氧化酶活性, 丙二醛

Abstract:

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.