鳗鲡病原菌外膜蛋白三联表达载体的构建、表达与表达产物的纯化  

Construction,expression and purification of triple expression vector of outer membrane protein of three pathogens isolated from diseased eels

作　　者：赵金平[1] 段丽华[1] 郭松林[1] 

机构地区：[1]集美大学水产学院/鳗鲡现代产业技术教育部工程研究中心,厦门361021

出　　处：《华中农业大学学报》2015年第3期104-110,共7页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31001136);鳗鲡现代产业技术教育部工程研究中心开放课题(RE201506)

摘　　要：为研制能同时预防鳗鲡3种常见病原菌感染的三联外膜蛋白疫苗,分别选择创伤弧菌(Vibrio vulnificus)、迟缓爱德华氏菌(Edwardsiella tarda)和嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白的ompU、ompA和omp porinП基因,设计特异性引物后分别扩增基因中表达外膜蛋白膜外区域且抗原决定簇丰富的3个基因片段并通过融合PCR技术进行连接。根据表达载体(pGEX-2T-his)的限制性酶切位点,在连接片段的两端引入BamHⅠ和EcoRⅠ两个酶切位点,将3个外膜蛋白基因片段与质粒连接后成功构建了重组表达载体。重组表达载体成功转化大肠杆菌(E.coli BL21)后,当菌液浓度(D600nm)为0.8时加入0.25mmol/L IPTG,16℃培养过夜后得到了高效表达。表达产物用镍柱进行洗脱纯化后经梯度尿素复性获得了分子质量约85ku的高纯度蛋白。In order to acquire vaccine antigen of triple outer membrane protein （omp） which can simultaneously prevent three common pathogens in eel, three genes of ompU, ornpA and ornp porin -Π were selected from Vibrio vulnificus, Edwardsiella tarda and Aeromonas hydrophila, respectively. Three gene fragments which encoded the outer regions of the three omps and showed rich epitopes were amplified and combined by two times of ＂two-steps＂ fusion PCR. According to the restriction sites of the expression vector （pGEX-2T-his plasmid）, two restriction sites of BamH I and EcoR I were added to the 5＇ points of two primers which are used to amplify fragments of ornpU and omp porin Π, respectively. The fusion fragment was connected with pGEX-2T-his plasmid and the recombinant expression vector which expressed the fusion omp was constructed. After the vector was transformed into E. coli BL21, the fusion omp with a molecular weight of 85 ku was highly expressed by using 0. 25 mmol/L IPTG to in- duce the tansformant （E. coli BL21） overnight at 16℃ in a suitable bacteria concentration （D600nm= 0.8）. The expressed protein was eluted and purified by with gradient of urea. nickel column, and then renaturated by dialysis

关 键 词：迟缓爱德华氏菌 创伤弧菌 嗜水气单胞菌 三联外膜蛋白 表达与纯化 

分 类 号：S942.5[农业科学—水产养殖]