GST-hCAP融合蛋白表达载体的构建及其在原核细胞中的表达-东南大学学报医学版

GST-hCAP融合蛋白表达载体的构建及其在原核细胞中的表达

单位：1. 中国医科大学附属盛京医院骨科, 辽宁沈阳 110004;
2. 中国医科大学基础医学院细胞生物学教研室,细胞生物学卫生部重点实验室,辽宁沈阳 110001;
3. 明尼苏达大学实验病理系,美国明尼苏达州明尼艾波利斯 55455

分类号：Q257; R-33

出版年·卷·期（页码）：2012·31·第一期（24-28）

摘要：

目的:构建GST标签的人c-Cbl相关蛋白(GST-hCAP)融合蛋白表达载体,并在大肠埃希菌(E.coli)中诱导其表达。方法:从COS-1细胞中提取mRNA,反转录为cDNA。用PCR方法扩增出hCAP基因全长,通过EcoRⅠ和XhoⅠ酶切位点将其定向插入pGEX-5X-1载体中,构建原核表达质粒pGEX-5X-1-hCAP,并转化E.coli DH5α,通过限制性内切酶酶切电泳和DNA测序鉴定正确后,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,原核表达质粒GST-hCAP构建成功,用Western blot方法也证实了GST-hCAP融合蛋白的表达。结论:成功地构建了GST-hCAP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CAP及研究其结构与功能提供了前提基础。

Objective: To construct GST-tagged human c-Cbl-associated protein(GST-hCAP) fusion protein expression vector and induce its expression in Escherichia coli(E.coli). Methods: Total mRNA was extracted from COS-1 cells and cDNA was synthesized by reverse transcription. The hCAP coding sequence was amplified by polymerase chain reaction(PCR) and subcloned into pGEX-5X-1 vector. The insert was identified by restriction enzyme digestion and DNA sequencing. pGEX-5X-1-hCAP and pGEX-5X-1 were transformed into E.coli BL21, induced by IPTG and identified by SDS-PAGE and Western blot. Results: The prokaryotic expression plasmid pGEX-5X-1-hCAP was successfully constructed and confirmed by enzyme digestion and sequencing. The GST-hCAP fusion proteins were identified by Western blot. Conclusion: The prokaryotic expression plasmid of hCAP 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 CAP protein and the biological function of CAP.

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