人抗黏病毒蛋白-MxA基因克隆及在大肠杆菌中的表达

目的克隆人抗黏病毒蛋白-MxA基因,构建其原核表达载体并进行表达、纯化、活性鉴定,为深入开展该蛋白功能的研究奠定基础。方法从IFN-β诱导的A549细胞系中提取总RNA,通过RT-PCR技术扩增出MxA全长cDNA,克隆于pUC119载体,酶切鉴定后并经测序证实序列正确,再克隆于pET-32a（＋）载体上构建表达载体pET-32a（＋）-MxA,转化大肠杆菌BL21,经IPTG诱导表达并优化诱导条件,通过NTA-Agarose亲和层析和割胶电洗脱法获取纯化MxA蛋白,用SDS-PAGE电泳分析表达产物,并通过对病毒VSV的抑制作用鉴定其活性。结果琼脂糖凝胶电泳结果显示RT-PCR扩增出两条片段,大小同预计片段相符,并经测序证实与公布的MxA基因序列一致;重组载体转化菌经IPTG诱导表达,SDS-PAGE电泳初步鉴定,目的蛋白约占总菌体蛋白的25%,与预计的分子量91kd相一致。用NTA-Agarose亲和层析法可获得纯度〉95%的目的蛋白,割胶电洗脱回收法可获得纯度〉90%的目的蛋白。结论克隆了人抗黏病毒蛋白-MxA基因,克隆基因在大肠杆菌中获得了较高水平的表达,并通过两种方法获得了该蛋白的纯化物。

Cloning and Expression of Human Myxovirus Resistance A Gene MxA in E.coli

Objective To obtain human myxovirus resistant MxA cDNA, express and purify the protein for further study of its function in vitro. Methods The cDNA of MxA was amplified with the mRNA templates from the INF-β-induced A549 cells by RT-PCR. The recombinant expression vector pET-32a（＋）-MxA was constructed and transformed into E.coli BL21. The fusion protein was induced with IPTG under various conditions and isolated by NTA-agarose resin and electroelution respectively, and identified in 12% SDS-PAGE. Results The RT-PCR products in agarose electrophoresis gel showed the expected DAN bands. The cloned MxA gene had the same nucleotide sequences as that from GENE Bank. Twnty-five percent MxA of the total cell proteins, having the predicted 91 kd molecule weight, was estimated in the IPTG-induced expression of BL21 transformed with pET-32a （＋）-MxA by electrophoresis of SDS-PAGE gel. Ninety-five percent purity of the protein was obtained by NTA-agarose resin, and 90% by electroelution. Conclusion The cDNA is successfully cloned. The MxA protein can be highly expressed in BL21 and purified. The purified protein is useful in later use.