猪轮状病毒vp4基因在大肠杆菌中的表达  

Expression of vp4 Gene from Porcine Rotavirus in E. coli

作　　者：宋岩[1] 李一经[1] 魏丽丽[1] 于小龙[1] 樊琛[1] 师东方[1] 

机构地区：[1]东北农业大学动医学院病原分子生物学实验室,黑龙江哈尔滨150030

出　　处：《中国病毒学》2004年第5期462-466,共5页Virologica Sinica

基　　金：黑龙江省十五攻关课题(GC01B510)

摘　　要：以猪轮状病毒JL94株核酸为模板扩增该病毒vp4全基因,对扩增产物进行测序及序列比较;根据VP4的5’端(1bp～750bp)特异片段主要决定其活性的观点,再设计一对引物扩增该主要抗原位点基因,将此主要抗原位点基因同pGEX-6P-1载体连接并转化入E.coli.BL21(DE3)plays,经IPTG诱导表达出蛋白质;对表达的蛋白进行Westernblot分析、纯化和血清中和抗体试验。结果表明:JL94株与国外分离株CRW-8株、BEN-307株vp4全基因片段氨基酸同源性分别为96.98%和98.05%,说明JL94株与CRW-8株、BEN-307株属于同一VP4血清型;经IPTG诱导VP4主要抗原位点基因获得了高效表达,表达量占菌体蛋白的26%;Westernblot结果和所表达的融合蛋白免疫小鼠产生的中和抗体能阻断JL94在MA104细胞上引起的细胞病变,说明所表达蛋白有良好的生物学活性。A pair of primers was used in cloning and sequence analysis of vp4 gene from Porcine rotavirus JL94 isolated in China. According to the idea that 5’segments of VP4 (1bp～750bp) principally control the activity of vp4 gene, a pair of another primers was used in cloning and obtained major antigen sites. The gene was cloned into the expression plasmid pGEX-6P-1. The recombinant plasmid VP4-pGEX-6P-1 was transformed into E. coli BL21(DE3)plays and induced with IPTG. Homology of amino acids between CRW-8 strain and BEN-307 strain is 96.98% and 98.05% respectively. The product of the VP4 gene was 26% of total bacterial protein of BL21. Western blot test and neutralization test circumstantiate the protein of expression has biological activity.

关 键 词：猪轮状病毒 VP4基因 VP4主要抗原位点基因 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]