猪轮状病毒地方株JL94株VP6基因的克隆及其在大肠杆菌中的表达

以猪轮状病毒地方分离株JL94株病毒核酸为模板,通过RT-PCR扩增内衣壳蛋白基因VP6 cDNA,将其插入克隆载体质粒pMD18-T,并进行测序。从T载体上将VP6基因亚克隆到表达载体质粒pET-30a,转化大肠杆菌BL21(DE3),IPTG诱导表达,并利用Ni^2 金属螯合亲合层析纯化融合蛋白。结果表明,VP6基因全长1356bp,并在大肠杆菌中获得了高效表达,表达量可占菌体蛋白的26．5％,在表达的蛋白中,除45ku主要蛋白外,还有几条分子量较小的蛋白表达出来,这些融合蛋白经纯化后均具有良好的反应特异性。

Cloning and Expression of VP6 Gene of Porcine Rotavirus JL94 in Escherichia Coli

The cDNA segments of complete gene 6 of porcine rotavirus JL94 isolate were obtained by RT-PCR that using dsRNA extracted from the virus as template, then were cloned into pMD18-T vector and sequenced. The cloned gene was subcloned from recombinant plasmid pMD18-T-VP6 into expression plasmid pET-30a. The recombinant plasmid pET-30a-VP6 was transformed into E.coli BL21(DE_3)and induced with IPTG. The expressed products were purified by metal (Ni~(2+)) chelation affinity chromatography. The results of sequencing showed that JL94 isolate complete gene 6 cDNA was 1 356bp and was expressed effectively as fused protein in Escherichia coli. The product of the VP6 gene amounted to 26.5% of total bacterial protein of BL21, not only a 45ku protein band but also several smaller polypeptide bands were observed. The purified protein proved to be antigenic by Western-blot analysis using serum against JL94 rotavirus.