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目的构建重组高尔基体蛋白的原核表达质粒并在大肠杆菌中诱导表达,为进一步研发肝癌诊断试剂奠定基础。方法通过RT-PCR获得GP73目的片段,将其克隆入表达载体p ET-28a,经酶切及测序鉴定后,在BL21菌株中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,用Western Blot鉴定。结果扩增的片段大小与预期一致;p ET-28a-GP73重组质粒通过酶切鉴定与DNA测序证实序列正确;诱导的蛋白用SDS-PAGE电泳和Western Blot检测,与预期相符可见一特异性条带。结论成功获得GP73目的基因并构建了p ET-28aGP73原核表达载体,实现了该融合蛋白的可溶性表达。
Objective To construct a prokaryotic expression plasmid of recombinant Golgi protein and induce it in E.coli to lay the foundation for the further development of diagnostic reagents for hepatocellular carcinoma. Methods The target fragment of GP73 was obtained by RT-PCR and cloned into the expression vector p ET-28a. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21 strain with IPTG (isopropyl β-D-thiogalactoside) Induced expression, identified by Western Blot. Results The size of amplified fragment was as expected. The recombinant plasmid p ET-28a-GP73 was verified by restriction enzyme digestion and DNA sequencing. The induced protein was detected by SDS-PAGE and Western Blot. As expected, a specific fragment band. Conclusion The GP73 gene was successfully obtained and the prokaryotic expression vector p ET-28aGP73 was constructed. The fusion protein was expressed successfully.