HE4融合蛋白基因的构建、表达及鉴定

Construction,Expression of HE4 Fusion Protein and its identification

目的重组及表达人附睾上皮分泌蛋白4(HE4)的融合蛋白,为建立一种新的卵巢癌的诊断方法奠定基础。方法提取人卵巢癌细胞株SKOV3中总RNA,应用RT-PCR技术扩增出HE4基因,将其与PUCm-T载体连接,转化入TOP10菌中进行克隆并测序鉴定。进一步构建PET28a-HE4表达体系,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导,最终在BL21菌中表达HE4融合蛋白(含有His-Tag),并进行SDS-PAGE及western blot鉴定。结果SKOV3细胞株提取的RNA经RT-PCR扩增后得到一条400bp左右的DNA片断,经测序鉴定含HE4基因序列;成功构建了HE4融合蛋白的重组表达质粒;并在大肠杆菌中实现了可溶性高表达,经western blot鉴定为HE4融合蛋白。结论采用原核表达方法可获得高纯度的HE4融合蛋白,为进一步开发HE4诊断试剂打下基础,为卵巢癌的诊断开辟新途径。

Objective To clone human epididymis protein 4 （HE4） gene, construct its expression vector, and obtain HE4 fusion protein applied in diagnosis of the early ovarian carcinomas.Methods The gene encoding HE4 was cloned using RT-PCR technique from total RNA of ovarian carcinomas cells SKOV3, and the amplified gene was connected with PUCm-T vector and subcloned into TOP10.The recombinant plasmid PET28a-HE4 was constructed and transformed into E. coli BL21 cells, and protein expression （including His-Tag） was induced by IPTG and identified by SDS-PAGE and western blot. Results A band of DNA about 400 bp was continued as the HE4 gene by DNA sequence analysis, the expression system was constructed, and soluble expression of the HE4 fusion protein was obtained successfully and identified by western blot. Conclusion The fusion protein HE4 with high purity could he engineored by expressed in prokaryotic system that lay a foundation for developing the In Vitro diagnostic kit. The study has a good propects aiding for diagnosis in ovarian carcinomas.