创伤弧菌OmpU与嗜水气单胞菌OmpⅡ外膜蛋白二联表达及其初步免疫原性  

Combined Expression and Primary Immunogenicity Study of Outer Membrane Protein OmpU and OmpⅡ of Vibrio vnlnificus and Aeromonas hydrophila

作　　者：郭松林[1,2] 陆盼盼 冯建军[1,2] 林鹏[1] 

机构地区：[1]集美大学水产学院,福建厦门361021 [2]鳗鲡现代产业技术教育部工程研究中心,福建厦门361021

出　　处：《集美大学学报（自然科学版）》2017年第3期1-7,共7页Journal of Jimei University：Natural Science

基　　金：国家自然科学基金项目(31001136);福建省自然科学基金项目(2015J01143);福建省教育厅项目(JA15283)

摘　　要：从发病的养殖鳗鲡中分离出创伤弧菌(Vibrio vulnificus)和嗜水气单胞菌(Aeromona shydrophila),提取其基因组DNA,再通过PCR法克隆创伤弧菌外膜蛋白Omp U和嗜水气单胞菌外膜蛋白OmpⅡ的基因全长。采用融合PCR法体外连接这2个基因表达膜外片段的序列并成功构建了二联表达载体(p GEX-2T-Vibr-Aero-his)。在大肠杆菌(BL21)的吸光度A_(600)为0.6~1.0时,采用1.0 mmol/L IPTG诱导剂,16℃过夜诱导表达。表达产物经离心柱亲和层析纯化后获得分子量为82.2 ku的外膜蛋白。蛋白经透析复性后免疫于鳗鲡以测定其血清抗体效价。结果表明,重组蛋白注射组的鳗鲡血清抗体效价在免疫后第14天和第21天显著(P<0.05)高于PBS对照组,第28天和第42天达到极显著(P<0.01)水平。In this study, template DNAs were extracted from pathogenic Vibrio vulnificus and Aeromonas isolated from diseased eels and confirmed by challenge experiment. According to gene sequences coding the protein Omp in GenBank database,two pairs of primers were designed respectively and the whole gene fragments of OmpH of A. hydrophila and OmpU of V. vulnificus were amplified by PCR. Two gene frag-ments ,encoding the out part of the membrane region of OmpU and OmpH respectively, were successfully ob-tained and the recombinant expression vector （ pGEX - 2T - Vibr - Aero - his） was combined by fusion PCR. Expression of the bivalent Omp was induced over night by 1. 0 mmol/L IPTG at 16 ^ when the concentration of 600 nm optical density of the bacteria was 0. 6 - 1.0 , and the expressed protein with the molecular weight of 82. 2 ku was obtained and purified by HisPur Ni - NTA Resin and Kits. Eels were immunized with the b i-valent OMP after purified and renatured by dialysis to determine the antibody tite r. Compared with eels inPBS group, the serum titers of anti-F. vulnificus and A. hydrophila antibody in eels of Omp group , showed sig-nificant （P 〈 0. 05） increase on 14 d and 21 d , and the different between the two groups reached very signifi-cant levels （ P 〈 0. 01） on 28 d and 42 d. The study laid a foundation for the vaccine research and applica- tionin aquaculture of the bivalent outer membrane protein.

关 键 词：创伤弧菌 迟缓爱德华氏菌 OmpU OmpⅡ 表达产物 免疫原性 

分 类 号：S917[农业科学—水产科学]