sMICA基因的克隆表达及其纯化

Cloning and expression of MHC class Ⅰ chain-related A gene in E.coli

目的构建重组MICA原核表达载体并探讨MICA对NK细胞毒效应的影响。方法经RT-PCR从Hela细胞获取MICA基因,经适当酶切后构建表达载体pBV220-MICA,转入大肠杆菌DH5α进行表达。重组MICA蛋白经纯化后与NK92细胞孵育观察对NK细胞毒效应的影响。结果带有重组质粒pBV220-MICA的大肠杆菌经热诱导后,以可溶性形式表达重组MICA蛋白,纯化后的重组MICA蛋白纯度为95%。经重组MICA处理的NK92细胞对MICA表达阳性的肿瘤细胞的杀伤作用明显降低。结论构建了pBV220-MICA重组质粒,并在大肠杆菌中获得可溶性表达,为研究MICA与NK细胞受体的相互作用机制奠定了基础。

Purpose To obtain recombinant MICA expression vector and to investigate its effects on cytotoxic activity of natural killer cells. Methods MICA cDNA fragments were amplified from Hela cells by RTPCR method . After digestion by restriction enzyme, the MICA gene fragment was cloned into prokaryotic expression vector pBV220 and the recombinant MICA expression vector was constructed and expressed in host E. coli DH5 α. The recombinant protein was purified by chromatography and its effects on cytotoxic activity of natural killer cells were studied. Results the recombinant MICA was expressed in soluble form, and the expression level is about 20% of total bacteria proteins. After purification, the target protein could be obtained with the purity of 95 %. The cytotoxity of NK92 to MICA positive tumors reduced significantly after being cocuhured with recombinant MICA. Conclusion The results demonstrated that MICA expression vector was constructed successfully and rMICA could be expressed in soluble form. The recombinant MICA was applicable in exploring the interation between MICA and natural killer cell receptors.