一个与大鲵蛙病毒(ADRV)感染相关基因-6R的克隆、原核表达及其产物分离  

Cloning, prokaryotic expression and protein separation of 6R, an Andrias davidianus ranaviurs(ADRV) infection-related gene

作　　者：李涛[1,2] 陈中元[1,2] 张奇亚[1,2] 

机构地区：[1]中国科学院水生生物研究所/淡水生态与生物技术国家重点实验室,湖北武汉430072 [2]中国科学院大学,北京100049

出　　处：《中国水产科学》2015年第2期353-358,共6页Journal of Fishery Sciences of China

基　　金：国家科技支撑计划课题(2012BAD25B02)

摘　　要：在新近报道大鲵蛙病毒(Andriasda vidianus ranaviurs,ADRV)基因组的基础上,我们对ADRV 6R进行了克隆、原核表达及其产物的分离,这是一个经生物信息分析表明与病毒感染相关的功能基因。先经PCR扩增长到711 bp的ADRV6R,构建含扩增片段的克隆质粒p MD18-T-6R。再构建原核表达质粒p ET-32a-6R,进行测序,并与水生动物蛙病毒相关基因同源序列进行比对,该扩增片段与预期一致,其编码分子量约为27 k D、含236个氨基酸的蛋白,与水产动物病毒US22蛋白家族(US22 family protein)成员有很高的同源性。然后对p ET-32a-6R进行转化、诱导表达,并利用Ni-NTA His-Bind亲和层析及不同浓度咪唑洗脱液对表达产物进行分离、电泳分析,结果显示,获得与预测分子量相符的45 k D融合蛋白(27 k D目的蛋白与18 k D His标签)。这为后续制备抗体、并开展大鲵蛙病毒与宿主的相互作用研究提供了有用的实验材料。In this study, the 6R gene, which may be a functional gene involved in Andrias davidianus ranaviurs（ADRV） infection by bioinformatics analysis, was cloned and expressed. ADRV 6R about 711 bp was amplified by PCR, cloned into plasmid p MD18-T and the cloning plasmid containing ADRV 6R gene（p MD18-T-6R） was constructed. Then, the recombinant prokaryotic expression plasmid p ET-32a-6R was constructed and sequenced. ADRV 6R gene was blasted with homologous sequences from other rana viruses of aquatic animals. The gene encodes a protein of 236 aa with a predicted molecular mass of 27 k D. Amino acid sequence alignment of ADRV 6R showed a high homology with US22 family protein members in the viruses of aquaculture animals. The recombinant prokaryotic expression plasmid p ET-32a-6R was transformed into DE3 and induced by IPTG. The expressed protein was purified with Ni-NTA His-Bind affinity chromatography, separated by different concentrations of imidazole eluent and analyzed by SDS-PAGE. A 45 k D fusion proteincontaining 27 k D interest protein and 18 k D his-tagged was obtained in accordance with the predicted molecular weight. These results provide useful experimental material for antibody preparation and study on ADRV-host interactions.

关 键 词：大鲵蛙病毒(ADRV) ADRV 6R 感染相关基因 US22蛋白家族 原核表达 蛋白分离 

分 类 号：S94[农业科学—水产养殖]