小鼠巨噬细胞炎性蛋白-1β和Ret蛋白融合基因的克隆、表达与纯化

Expression and purification of the fusion protein of mouse macrophage inflammatory protein 1βand Ret

目的构建小鼠巨噬细胞炎性蛋白-1β(murinemacrophageinflammatoryprotein1β,mMIP-1β)与原癌基因RET的融合基因并构建融合基因mMIP-RET的原核表达载体,在大肠杆菌中进行表达。方法用RT-PCR方法分别扩增mMIP1β和RET的基因片段,利用分子克隆的方法将这两个片段用铰链GGIPG连接,并克隆到表达载体pET22b中。双酶切且测序正确后转化大肠杆菌BL21,构建表达pET22bmMIP1β-RET/His融合蛋白的菌株。经IPTG诱导表达后,ProBondResin进行亲和层析纯化,然后纯化产物进行复性及超滤浓缩。结果成功克隆mMIP1β和RET基因片段;成功地构建了pET22bmMIP1β-RET/His融合蛋白原核表达载体,在大肠杆菌中获得表达,并对表达产物进行了纯化,和Westernblot鉴定。结论成功制备高纯度mMIP1β-Ret/His融合蛋白,为进一步研究修饰的肿瘤抗原的生物学功能奠定了基础。

Objective To produce the mMIP/Ret fusion protein in Escherichia coli. Methods The cDNA encoding mMIP1β/RET was amplified by using RT-PCR method and cloned into pET22b vector. After identification by restriction endonuclease digestion and sequencing, the expression plasmid was introduced into E. coli strain BL21 （DE3）. The expression of the His6-tagged fusion protein in the genetically engineered bacteria was induced by IPTG, and the expression products was purified by ProBond Resin system. Results The mMIP and RET genes were cloned and the pET22b mMIP1β-RET/His prokaryotic expression plasmid was constructed and expressed successfully. The expression products were purified and identified by Western blotting. Conclusion The fusion mMIP1β-REF/His protein is expressed and purified successfully. These results provide essential conditions for further research on the biological function of mMIP1β-modified tumor antigen Ret.