东亚三角涡虫肌球蛋白轻链(MLC)融合蛋白原核表达载体的构建及其在大肠杆菌中的表达

Construction of Prokaryotic Expression Vector of Planarian Myosin Light Chain and Fusion Protein Expression in Escherichia Coli

构建了涡虫肌球蛋白轻链融合蛋白原核表达载体,并进行表达,根据涡虫基因文库中肌球蛋白轻链(MIc)基因完整的ORF序列设计合成特异性引物,通过PCR扩增涡虫MIc基因,并插入到融合蛋白原核表达载体PET-28a中,转化宿主菌BL21(DE3)细胞,0.4mMol/L的IPTG诱导表达MLC蛋白.重组质粒测序和酶切结果显示Mlc基因已正确插入PET-28a中,重组蛋白经SDSPAGE在18.2KD处有一条明显的蛋白表达条带。western blot检测得到同样大小的条带。结果表明,涡虫His-MLC融合蛋白已成功表达。

The prokaryotic expression vector of planarian myosin light chain （Mlc） is constructed. The fusion protein is expressed in Escherichiacoli. Planarian Mlc cDNA is amplificated by RT-PCR which uses cDNA from the planarian genebank and special primers designed according to the known sequence of Mlc ORF. It＇s inserted into the prokaryotic expression plasmid PET-28 a vector and introduced into a strain of E. coli BL21 （ DE3 ）. The fusion protein expression is induced by IPTG 0. 4 mMol/L. The analysis results from recombinant plasmid DNA sequencing and restriction enzyme digestion show that Mlc gene had been correctly inserted PET-28a. The recombinant protein is purified by affinity purification,and its molecular mass was estimated to be approximately 18.2 KD by SDS-PAGE. Western blots with His antibody showed similar result. It＇s indicated that the His-MLC fusion protein of planarian is successfully expressed.