GST-hMunc18-1融合蛋白表达载体的构建及其在原核细胞中的表达

Construction of expressing vector of GST-hMunc18-1 prokaryotic plasmid and expression in the prokaryocyte

目的:构建hMunc18-1的原核表达载体并诱导、纯化和鉴定其表达。方法:从人HEK293细胞中提取mRNA,反转录为cDNA。用PCR方法扩增出hMunc18-1基因全长,通过EcoRⅠ和XhoⅠ酶切位点将其定向插入pGEX-6p-1载体中,构建原核表达质粒pGEX-6p-1-hMunc18-1。异丙基β-D硫代半乳糖苷(IPTG)在E.coli BL21大肠杆菌中诱导GST-hMunc18-1融合蛋白表达,利用Glutathione Sepharose 4B纯化诱导的融合蛋白,并经Western Blot鉴定结果。结果:hMunc18-1编码序列克隆至pGEX-6p-1载体中,双酶切鉴定片段大小为1785 bp,在E.coli BL21中IPTG诱导融合蛋白的表达,分子质量约为67 000 Da,成功纯化出GST及GST-hMunc18-1蛋白,Western Blot检测到蛋白表达。结论:成功地构建了hMunc18-1基因原核表达载体,证实了其在原核细胞大肠埃希菌中的表达,为进一步纯化Munc18-1及研究其结构与功能奠定了基础。

Objective： To construct prokaryotic expression vector of human Muncl 8-1 gene and induce, purify and iden-tify its recombinant protein expression. Methods： Total mRNA was extracted from HEK293 cells and eDNA was synthe-sized by reverse transcription. The hMuncl8-1 coding sequence was amplified by polymerase chain reaction （PCR） and subcloned into pGEX-6p-1 vector. The insert was identified by restriction enzyme digestion and DNA sequencing, pGEX-6p-l-hMuncl8-1 and pGEX-6p-1 were transformed into E. coli BL21, induced by IPTG and identified by SDS-PAGE and Western Blot. Results： The coding sequence of hMunel8-1 gene was cloned into pGEX-6p-lvector, which was trans- formed into E. coli BL21. The length of fragment was 1785 bp. The expression of GST- hMuncl8-1 fusion protein was in- duced by IPTG, and the molecular weight of protein was 67 000 Da. GST protein and GST- hMuncl8-1 fusion protein were purified successfully and detected by Western Blot. Conclusion： The prokaryotic expression plasmid of hMuncl8-1 is successfully constructed and the expression of fusion proteins in E. coli is confirmed. This study provides the basis for the further research on purifying Muncl8-1 protein, the structure and the biological function of Muncl8-1.