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  

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

作　　者：陈淑红[1] 李一经[1] 师东方[1] 

机构地区：[1]东北农业大学动物医学院,哈尔滨150030

出　　处：《畜牧兽医学报》2004年第4期443-448,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：黑龙江省"十五"攻关项目(GC01B510)

摘　　要：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VP6cDNA,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Ni2+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。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.

关 键 词：猪 轮状病毒 JL94株 VP6基因 基因克隆 大肠杆菌 基因表达 

分 类 号：S852.659.4[农业科学—基础兽医学] S858.28[农业科学—兽医学]