妊娠相关血浆蛋白A的原核表达及蛋白纯化  

Prokaryotic expression and purification of pregnancy-associated plasma protein A

作　　者：曾昭伟[1] 常艳敏[1] 王学谦[1] 

机构地区：[1]天津市南开医院检验科,天津300100

出　　处：《临床检验杂志》2016年第3期186-189,共4页Chinese Journal of Clinical Laboratory Science

基　　金：天津市卫生局科技基金(2013KY24)

摘　　要：目的构建原核表达PAPP-A重组蛋白,并对蛋白质进行纯化。方法根据DNAstar软件分析获得的编码PAPP-A特异抗原表位,利用Primer Premier 5.0软件设计引物,以PAPP-A c DNA-质粒转化菌液为模板,进行PCR扩增。PAPP-A的DNA和p ET42a质粒分别双酶切,经琼脂糖凝胶电泳及胶回收后进行连接反应,转化至大肠埃希菌Top10,筛选阳性克隆经测序鉴定正确后,转化至大肠埃希菌BL21,诱导产生PAPP-A重组蛋白,利用镍柱初步纯化及离子交换层析柱进一步纯化,用SDS-PAGE及DEAE层析鉴定重组抗原蛋白。结果筛选出抗原表位集中区,在大肠埃希菌BL21中高效表达了重组的PAPP-A蛋白,经过镍柱及离子交换层析纯化后,PAPP-A浓度为0.22 g/L,DEAE层析分析证实其纯度较高。结论成功筛选出PAPP-A的抗原表位集中区,并制备了重组抗原蛋白,为PAPP-A的后续临床研究提供依据。Objective To prepare and purify recombinant pregnancy-associated plasma protein A（ PAPP-A） with prokaryotic expression system. Methods The nucleic acid sequence encoding the epitope of recombinant PAPP-A was obtained by the analysis of DNAstar software,and was amplified by PCR with the primers designed by Primer Premier 5. 0 software,and the PAPP-A c DNA plasmid as template. Then,the obtained PAPP-A DNA and plasmid p ET42 a were digested with enzymes,and connected after purification by agarose gel electrophoresis. The obtained recombinant plasmid was transfected into E. coli TOP10. After the recombinant plasmid was identified by sequencing,it was transfected into E. coli BL21,and was induced to express recombinant PAPP-A protein,which was further purified by nickel column and ion exchange chromatography,and identified by SDS-PAGE. Results The nucleic acid sequence encoding the epitopes of PAPP-A was screened successfully,and the constructed recombinant plasmid expressed the recombinant PAPP-A protein efficiently in E. coli BL21. After the recombinant protein was purified by nickel column and ion exchange chromatography,its concentration could reach to 0. 22 g / L,and the high purity could be confirmed by DEAE sephacel. Conclusion The main epitopes of PAPP-A are screened successfull,and the recombinant PAPP-A protein is prepared efficiently,which provides the evidence for further establishing the detection method of PAPP-A.

关 键 词：妊娠相关血浆蛋白A 抗原表位 原核表达 

分 类 号：R392.33[医药卫生—免疫学]