卵巢癌肿瘤标记物HE4基因的分子克隆与蛋白表达

目的构建HE4基因及蛋白表达系统,用于卵巢癌的早期诊断。方法提取卵巢癌细胞H08910的总RNA,利用RT-PCR技术扩增HE4基因,然后将该片段进行T-A克隆并测序鉴定,纯化回收后亚克隆于原核表达载体PGEX-4T-1中,然后提取质粒,酶切鉴定,最后经IPTG诱导,原核表达重组子在TOP10宿主菌进行表达,并进行SDS-PAGE、WesternBlot分析与鉴定。结果应用RT-PCR技术,从卵巢癌HO8910的总RNA中扩增到一条大小约为400bp的片段,经T-A克隆与测序鉴定,证实此片段为HE4基因,将PGEX-4T-1重组子转化入宿主菌,用IPTG诱导菌体表达HE4重组蛋白,经SDS-PAGE电泳和WesternBlot鉴定证实,在相对分子量约39000处可见明显的特异性的高表达条带。结论已成功克隆HE4基因,并经IPTG诱导在TOP10宿主菌中表达了相应的蛋白,为进一步制备卵巢癌的早期诊断试剂奠定基础。

Cloning and Expression of HE4 Gene from Human Ovarian Carcinomas

Objective In order to detect the early ovarian carcinomas,we cloned the HE4 gene and constructed its expression vector.Methods The HE4 gene was amplified and cloned by RT-PCR and T-A clone techniques.The amplified HE4 gene was subcloned into expression vector PGEX-4T-1.Recombinant plasmids were identified by restriction endonuclease digestion and sequencing.The recombinant HE4 protein was expressed in TOP10 E.coli under the IPTG induction.Expressed protein was identified with Western blot analysis.Results A specific band of about 400 bp was amplified using RT-PCR from total RNA of ovarian carcinomas cells and confirmed as the HE4 gene via T-A clone and DNA sequencing analyses.SDS-PAGE showed a clear protein band with a molecular weight of 39 kDa in the IPTG-induced samples.The protein band was confirmed as HE4 protein by Western blot analysis.Conclusion The HE4 gene was successfully cloned from ovarian carcinomas cells and the HE4 protein was expressed in prokaryotic expression vector.