pGEX-5X-1-hLMO4原核质粒构建及重组蛋白表达-东南大学学报医学版

pGEX-5X-1-hLMO4原核质粒构建及重组蛋白表达

单位：1. 中国医科大学细胞生物学教研室, 卫生部细胞生物学重点实验室, 教育部医学细胞生物学重点实验室, 辽宁沈阳 110001;
2. 辽宁省人民医院消化内科, 辽宁沈阳 110016

分类号：Q291; Q591.2

出版年·卷·期（页码）：2012·31·第二期（139-143）

摘要：

目的:构建hLMO4的原核表达载体并诱导、纯化和鉴定其表达。方法:hLMO4全长编码基因经BamHⅠ和XhoⅠ双酶切后,克隆至GST融合表达载体pGEX-5X-1,异丙基β-D硫代半乳糖苷(IPTG)在BL21大肠杆菌中诱导GST-hLMO4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:hLMO4编码序列克隆至pGEX-5X-1载体中,双酶切鉴定片段大小为500 bp,在E.coli BL21中IPTG诱导融合蛋白的表达,分子质量约为49 000 Da,成功纯化出GST及GST-hLMO4蛋白,Western blot检测到蛋白表达。结论:构建了hLMO4基因原核表达载体,鉴定了GST-hLMO4融合蛋白表达。

Objective: To construct prokaryotic expression vector of human LMO4 gene and induce, purify and identify its recombinant protein expression. Methods: The hLMO4 coding sequence was digested with BamHⅠ and XhoⅠ enzymes, and cloned into pGEX-5X-1. The expression of GST-hLMO4 fusion protein was induced by IPTG and identified by Western blot. Results: The coding sequence of hLMO4 gene was cloned into the pGEX-5X-1 plasmid which was transformed into E.coli BL21. The length of fragment was 500 bp. The expression of GST-hLMO4 fusion protein was induced by IPTG, and the molecular weight of protein was 49 000 Da. Conclusion: The recombinant prokaryotic plasmid was successfully constructed into pGEX-5X-1. The expression of GST-LMO4 fusion protein was induced by IPTG and identified.

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