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

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.