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 cells, and cDNA was formed by reverse transcription. 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 transformed into E. coli BL21, induced by IPTC and identified by SDS-PAGE and Western blot. Results The prokaryotic expression plasmid pGEX-4T-2-hPlk2 was successfully constructed and confirmed by enzyme digestion and sequencing. The CST-hPlk2 fusion proteins were expressed and confirmed by Western blot. Conclusions The prokaryotic expression plasmid of hPlk2 was successfully constructed and the expression of fusion proteins in E. coli was confirmed. This study provides the basis for the further research on purifying Plk2 protein and the biological function of Plk2.%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BamH Ⅰ和Xho Ⅰ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Ecoli 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.
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