重组金属硫蛋白2A质粒的构建与表达

Construction and expression of recombinant plasmid of metallothionein 2A

目的构建重组金属硫蛋白2A(MT-2A)原核表达质粒,并在大肠埃希菌BL21(DE3)中表达,为进一步研究MT-2A奠定基础。方法从人骨骼肌细胞cDNA文库中PCR扩增MT-2A基因DNA序列,构建重组表达质粒MT2A-pET32a,PCR与DNA测序法鉴定插入序列;重组细菌用IPTG诱导表达,His-bind亲和层析柱纯化MT-2A蛋白,Western blotting鉴定蛋白特性。结果扩增获得的MT-2A基因片段长186bp,DNA测序证实MT2A-pET32a重组质粒构建正确;表达的融合蛋白相对分子质量约为29×103,Western blotting证实为目的蛋白。结论成功构建了重组原核表达质粒MT2A-pET32a,在大肠埃希菌内诱导表达并纯化获得MT-2A。

Objective To construct prokaryotic expression plasmid of metallothionein 2A （MT-2A） and induce the expression of constructed plasmid in E.coli BL21（DE3）.Methods The MT-2A cDNA was amplified by PCR from skeletal muscle library and inserted into expression vector pET32a.The recombinant plasmid MT2A-pET32a,which could express the fusion protein of MT-2A,was then transferred into E.coli BL21 （DE3）.The target protein was identified using SDS-PAGE.Affinity chromatography was used for protein purification.The immune reactivity of purified MT-2A was identified by Western blotting using anti-MT2A specific antibody.Results A gene fragment of 186 bp was acquired by PCR,and the constructed recombinant plasmid MT2A-Pet32a was confirmed by DNA sequencing.The expressed recombinant protein was with relative molecular weight of 29×103,and was identified to be target protein by Western blotting.Conclusion The recombinant plasmid MT2A-pET32a was constructed successfully and the fusion protein could be expressed in E.coli.