HE4两种融合蛋白的基因构建和原核表达  

Construction and expression of two kinds of HE4 fusion protein

作　　者：张雪峰[1,2] 刘一兵[1,2] 李子颖[1,2] 贾娟娟[1,3] 王斌[1,2] 韩世泉[1,3] 

机构地区：[1]原子高科股份有限公司,北京102413 [2]中国原子能科学研究院,北京102413 [3]中国食品药品检定研究院,北京100050

出　　处：《中国免疫学杂志》2014年第12期1666-1669,共4页Chinese Journal of Immunology

摘　　要：目的:构建人附睾蛋白4(HE4)的原核表达系统,纯化重组蛋白并检测其活性。方法:提取卵巢透明癌细胞总RNA,利用RT-PCR技术扩增HE4基因,分别构建pGEX-4T-1-HE4和PET26b-HE4两种原核表达系统。以异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,最后采用SDS-PAGE和HE4商品化免疫分析试剂盒对表达产物进行鉴定。结果:成功构建了两种HE4融合蛋白的重组表达质粒,表达产物经SDS-PAGE鉴定,分别在相对分子量约38 000处和12 000处见到表达条带,并可与HE4单克隆抗体特异性结合。结论:采用原核表达方法获得了HE4的两种融合蛋白,为下一步开发免疫分析诊断试剂奠定了基础。Objective：To express human epididymis protein 4 （HE4） in prokaryotic cells,purify the expressed product and de-termine its activity by immunoassay kit.Methods： The gene encoding HE 4 was cloned using RT-PCR technique from total RNA of ovarian carcinoma cells ES-2,the amplified HE4 gene was cloned into prokaryotic expression vector pGEX-4T-1 and PET26b respective-ly.The recombinant plasmid pGEX-4T-1-HE4 and PET26b-HE4 were constructed and transformed into E.coli BL21 cells respectively, and protein （ GST-HE4 and His-HE4 ） expressions were induced by IPTG and identified by SDS-PAGE and commercial ELISA kit.Results：Restriction analysis and sequencing proved that recombinant plasmid pGEX -4T-1-HE4 and PET26b-HE4 were constructed correctly.The expressed recombinant proteins ,with the relative molecular mass of about 38 000 and 12 000 ,showed specific binding to monoclonal antibody against HE 4.Conclusion：Two kinds of recombinant HE 4 protein are successfully expressed in prokaryotic cells , which laid a foundation of preparation of immunoassay reagents.

关 键 词：卵巢癌 人附睾蛋白4(HE4) 克隆 表达 

分 类 号：R730[医药卫生—肿瘤] Q789[医药卫生—临床医学]