人肌球蛋白调节性轻链的原核表达及纯化-东南大学学报医学版

人肌球蛋白调节性轻链的原核表达及纯化

单位：东南大学医学院病理学与病理生理学系, 江苏南京 210009

分类号：Q813

出版年·卷·期（页码）：2013·32·第四期（408-412）

摘要：

目的: 构建pGEX-MLC2重组质粒,在大肠杆菌中表达并纯化谷胱甘肽硫转移酶(GST)-MLC2融合蛋白,以用于MLC2抗体的制备。方法: 从人乳腺癌MCF-7细胞中提取总RNA,通过RT-PCR获得人MLC2 cDNA全长开放读码框架(open reading frame, ORF),并将其重组于原核蛋白表达质粒pGEX-6P-1中,经限制性内切酶和DNA测序鉴定,将该重组质粒转化大肠杆菌E.coli BL21,用异丙基β-D-硫代半乳糖苷(IPTG)诱导表达GST-MIC2融合蛋白后,进行Western blotting分析,鉴定GST-MLC2蛋白;通过Glutathione Sepharose 4B亲和层析柱纯化融合蛋白,并用SDS-PAGE电泳鉴定该融合蛋白的纯度。结果: 人乳腺癌MCF-7细胞MLC2的RT-PCR产物为535bp,符合预期;重组质粒pGEX-MLC2经BamHI和EcoRI双酶切出现特征性的525bp片段;DNA测序证实MLC2全长ORF序列正确无误。IPTG诱导表达的GST-MLC2融合蛋白分子质量约为46.6kDa,纯化后其蛋白纯度约95%,经Western blotting分析表明诱导表达的蛋白是GST-MLC2融合蛋白。结论: 人MLC2蛋白原核表达质粒的构建、GST-MLC2融合蛋白的表达和纯化,为MLC2抗体的制备及MLC2功能的深入研究奠定了基础。

Objective: To construct pGEX-MLC2 recombinant plasmid and to express and purify GST-MLC2 fusion protein. The fusion protein was prepared for MLC2 polyclonal antibody. Methods: The total RNA was extracted from human breast cancer cells MCF-7, the full-length open reading frame(ORF) of MLC2 cDNA was amplified by RT-PCR and then was cloned into the expression vector pGEX-6P-1. After identification by restriction enzyme digestion analysis and DNA sequencing, the recombinant clone was transformed into the competent cells E.coli BL21. GST-MLC2 fusion protein was induced expression by IPTG and was identified by Western blotting; Then the fusion protein was purified by Glutathione Sepharose 4B affinity chromatography and the purity was identified by SDA-PAGE electrophoresis. Results: The RT-PCR product of MLC2 was 535bp in line with expectations; The recombinant plasmid of pGEX-MLC2 was identified containing a distinctive 525bp fragment by BamH I and EcoR I double digestion; DNA sequencing confirmed that the MLC2 full-length ORF sequence was correct. The molecular weight of GST-MLC2 fusion protein by IPTG induced expression was about 46.6kDa and the purity of GST-MLC2 fusion protein was about 95% after purification. Western blotting analysis proved that the protein was GST-MLC2 fusion protein. Conclusion: The construction of prokaryotic expression plasmid for human MLC2 protein and the expression and purification of GST-MLC2 fusion protein lay the foundations for the preparation of MLC2 antibody and the further study on the function of MLC2.

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