GST-hPlk2融合蛋白表达载体的构建及其在原核细胞中的表达

Construction of GST-hPlk2 fusion protein expression vectors and the expression in prokaryotic cells

目的构建GST-hPlk2融合蛋白表达载体,并在原核细胞大肠埃希菌(E.coli)中诱导表达。方法从人HEK293细胞中提取mRNA,反转录为cDNA。用PCR方法扩增出hPlk2基因全长,通过BamHⅠ和XhoⅠ酶切位点将其定向插入pGEX-4T-2载体中,构建原核表达质粒pGEX-4T-2-hPlk2,并转化E.coli DH5α,筛选阳性重组子,通过限制性内切酶酶切电泳鉴定和DNA序列测定正确后,转入E.coli BL21中,经异丙基硫代β-D半乳糖苷大量诱导表达,SDS-PAGE电泳和Western blot鉴定。结果酶切电泳及测序结果证明,成功构建了原核表达质粒GST-hPlk2,并用Western blot方法证实了GST-hPlk2融合蛋白的表达。结论成功构建了GST-hPlk2原核表达载体,并证实了其在原核细胞E.coli中的表达,为进一步纯化Plk2及研究其结构与功能提供了前提基础。

Objective To construct GST-hPlk2 fusion protein expression vector and induce its expression in Esche-richia coli （E. coli）. Methods Total mRNA was extracted from HEK293 ceils, and cDNA was formed by reverse tran- scription. The hPlk2 coding sequence was amplified by polymerase chain reaction （PCR） and subcloned into pGEX-4T-2 vector. The positive recombinant was identified by restriction enzyme digestion and DNA sequencing. Then they were trans- formed into E. coli BI221, induced by IPTG and identified by SDS-PAGE and Western blot. Results The prokaryotic ex- pression plasmid pGEX-4T-2-hPlk2 was successfully constructed and confirmed by enzyme digestion and sequencing. The GST-hPlk2 fusion proteins were expressed and confirmed by Western blot. Conclusions The prokaryotic expression plas- mid of bPlk2 was successfully constructed and the expression of fusion proteins in E. coli was confirmed. This study pro- vides the basis for the further research on purifying Plk2 protein and the biological function of Plk2.