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目的构建p ET-28a-TK1融合基因表达载体,并诱导其在大肠杆菌中表达。方法提取人肝癌细胞(Hep G2)总RNA,应用RT-PCR、PCR技术扩增出TK1目的片段,将其与带有His标签的p ET-28a连接构建重组基因并测序鉴定;用异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白在BL21大肠杆菌中表达;最后通过SDS-PAGE及Western Blot鉴定。结果以Hep G2细胞的c DNA为模板,扩增出663bp的目的片段,将其克隆到p ET-28a原核表达载体,经酶切及DNA测序鉴定质粒构建正确;IPTG诱导后实现可溶性表达,经Western Blot鉴定为TK1融合蛋白。结论成功构建了TK1重组表达质粒,并在原核细胞中获得高效表达,为进一步开发TK1诊断试剂奠定基础。
Objective To construct p ET-28a-TK1 fusion gene expression vector and induce its expression in E. coli. Methods The total RNA of Hep G2 cells was extracted. The target fragment of TK1 was amplified by RT-PCR and PCR. The recombinant plasmid was ligated with pET-28a with His-tag. The recombinant plasmid was identified by sequencing. β-D-thiogalactopyranoside (IPTG) -induced recombinant protein was expressed in E. coli BL21; and finally identified by SDS-PAGE and Western Blot. Results The 663bp fragment was amplified from cp DNA of Hep G2 cells and cloned into p ET-28a prokaryotic expression vector. The plasmid was identified by restriction enzyme digestion and DNA sequencing. The soluble plasmid was induced by IPTG. Western Blot was identified as TK1 fusion protein. Conclusion The TK1 recombinant plasmid was successfully constructed and highly expressed in prokaryotic cells, which laid the foundation for the further development of TK1 diagnostic reagent.