创伤弧菌外膜蛋白OmpU的基因克隆与表达  

Gene Cloning and Expression of Outer Membrane Protein OmpU of Vibrio vulnificus

作　　者：李素一[1] 吴唯维[1,2] 李盼[1,3] 柯翎[1] 许斌福[1] 林晨韬[1] 林天龙[1] 陈叙[1] 

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003 [2]福建师范大学,福建福州350007 [3]福建农林大学,福建福州350002

出　　处：《福建农业学报》2013年第6期517-521,共5页Fujian Journal of Agricultural Sciences

基　　金：福建省农业科学院青年科技人才创新基金项目(2011QC-20);福建省农业科学院引进海外人才启动基金项目(HWRC2011-03);福建省自然科学基金项目(2011J05055);国家自然科学基金项目(31100658)

摘　　要：创伤弧菌是导致鳗鲡产生体表溃疡死亡的重要致病菌。本研究克隆了创伤弧菌FJ03-X2株外膜蛋白基因(ompU),构建含该基因的原核表达载体,命名为pET-32a-ompU,将其转化到大肠杆菌E.coli中进行融合表达。37℃,0.5mmol.L-1的IPTG诱导4h,融合蛋白rompU以可溶形式表达,采用Ni-NTA亲和层析法纯化融合蛋白,SDS-PAGE显示纯化后的rompU为与预期大小一致的单一条带。将纯化的rompU免疫SD级大鼠,ELISA检测收获的多克隆抗体血清效价,并通过Western-blot证实该OmpU多克隆抗体可以识别创伤弧菌FJ03-X2株中的天然外膜蛋白OmpU。Vibrio vulnificus is an important pathogen that causes the eel sepsis.In this study,the outer membrane protein U（ompU）gene of Vibrio vulnificus strain FJ03-X2is cloned,and then inserted into the prokaryotic expression vector,named pET32a-ompU.The plasmid was then transformed into E.coli strain to induce the expression of rompU protein.The soluble rompU protein can be expressed under the induction of IPTG（0.5mmol/ L）at 37℃for 4h.Then purified it by Ni-NTA His Binding affinity purification.SDS-PAGE displayed the purified rompU as a single band of the expected size.The purified rompU protein was then injected into SD level rats to produce polyclonal antibody.High specificity antiserum of FJ03-X2rompU is confirmed by ELISA detection.Western-blot confirmed that OmpU polyclonal antibodies can recognize the natural outer membrane protein OmpU in Vibrio vulnificus FJ03-X2.

关 键 词：创伤弧菌 外膜蛋白OmpU 克隆 原核表达 蛋白纯化 多克隆抗体 

分 类 号：S941[农业科学—水产养殖]