人肌球蛋白调节性轻链的原核表达及纯化

摘要

Objective: To construct pGEX-MLC2 recombinant plasmid and to express and purify GST-MLC2 fusion protein .The fusion protein was prepared for MLC 2 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 4 B affinity chromatography and the purity was identified by SDA-PAGE electrophoresis .Results: The RT-PCR product of MLC2 was 535 bp in line with expectations; The recombinant plasmid of pGEX-MLC2 was identified containing a distinctive 525 bp fragment by BamH I and EcoR I double digestion;DNA sequencing confirmed that the MLC 2 full-length ORF sequence was correct .The molecular weight of GST-MLC2 fusion protein by IPTG induced expression was about 46.6 kDa 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 MLC 2 protein and the expression and purification of GST-MLC2 fusion protein lay the foundations for the preparation of MLC 2 antibody and the further study on the function of MLC 2.%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535 bp，符合预期；重组质粒pGEX-MLC2经BamHI和EcoRI双酶切出现特征性的525 bp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46.6 kDa，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。