CAJ | 학술논문

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38000处和12000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。
목적：구건인부고단백4（ HE4）적원핵표체계통，순화중조단백병검측기활성。방법：제취란소투명암세포총RNA，이용RT-PCR기술확증HE4기인，분별구건pGEX-4T-1-HE4화PET26b-HE4량충원핵표체계통。이이병기-β-D-류대반유당감（ IPTG）유도중조단백표체，최후채용SDS-PAGE화HE4상품화면역분석시제합대표체산물진행감정。결과：성공구건료량충HE4융합단백적중조표체질립，표체산물경SDS-PAGE감정，분별재상대분자량약38000처화12000처견도표체조대，병가여HE4단극륭항체특이성결합。결론：채용원핵표체방법획득료HE4적량충융합단백，위하일보개발면역분석진단시제전정료기출。
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.