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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 hMunc18-1 and induce, purify and identify its expression. Methods: mRNA was extracted from human HEK293 cells and reverse transcribed into cDNA. The full length of hMunc18-1 gene was amplified by PCR and inserted into pGEX-6p-1 vector through EcoRⅠ and XhoⅠ restriction sites to construct prokaryotic expression plasmid pGEX-6p-1-hMunc18-1. The isopropyl β-D thiogalactoside (IPTG) induced the expression of GST-hMunc18-1 fusion protein in E. coli BL21 E.coli, purified the induced fusion protein with Glutathione Sepharose 4B, and identified the result by Western Blot. Results: The hMunc18-1 coding sequence was cloned into pGEX-6p-1 vector. The size of the fragment was 1785 bp. The recombinant protein was induced by IPTG in E.coli BL21. The molecular weight of the fusion protein was about 67 000 Da. GST and GST-hMunc18-1 protein were detected by Western Blot. CONCLUSION: The hMunc18-1 gene prokaryotic expression vector was successfully constructed and confirmed its expression in prokaryotic Escherichia coli, which laid the foundation for further purification of Munc18-1 and its structure and function.