猪源肠出血性大肠杆菌CD18株fedA基因的克隆及表达

Cloning and Expression of the fedA Gene of Enterohemorrhagic E.coli Strain CD18 Isolated from Pig in Chongqing

目的:获得重庆猪源肠出血性大肠杆菌(EHEC)强毒株CD18株fedA基因表达产物。方法:根据GenBank中的fedA基因序列设计引物,从重庆猪源EHEC强毒株CD18株基因组中经PCR扩增目的片段,克隆到pUC19质粒,亚克隆到表达载体pET28b(+)的SalⅠ-HindⅢ位点后转化大肠杆菌BL21(DE3),经IPTG诱导表达,Ni柱法纯化,SDS-PAGE及Western印迹检验表达产物。结果:CD18株fedA与文献报道的fedA的序列同源性为99.3%,推导的氨基酸序列的同源性为98.7%,表达产物在50～100mmol/L咪唑洗脱时出峰,SDS-PAGE显示其相对分子质量20000,Western印迹证明该蛋白条带能与分子标记蛋白His6抗体发生特异反应。结论:克隆到猪源EHECCD18株fedA基因,并在大肠杆菌中得到表达。

Objective： To clone and express the fedA gene from enterohemorrhagic E.coli（EHEC） strain CD18 isolated from edema disease of swine in Chongqing. Methods： A pair of the primer was designed according to the fedA gene sequence reported in GenBank. The fedA gene amplified from the genome DNA of CD18 strain by PCR was cloned to the pUC19 and sequenced, pET-28b（＋） was used to expressing fedA gene vector. The recombinant plasmid pET-28b（fedA） was then transoformed into E.coli BL21（DE3） and expression induced by IPTG. The protein of the fedA was purified by Ni-agrose gel. SDS-PAGE and Western-blotting were performed to analyze the fedA gene production. Results： Homology between the fedA gene of CD18 strain and the fedA gene reported in GenBank（Lot L26236） were 99.3% in DNA sequence and 98.7% in amino acid reduced by the DNA sequence. Ni-agrose gel affinity chromatogaph results showed the peak of the protein in the concentration of 50 to 100 mmol/L by mitrol elution. Mass weight of the products of the fedA is 20 kD by SDS-PAGE. Western-blotting results showed that the protein could reacted with antibaody against His6 protein. Conclusion： The fedA gene of enterohemorrhagic E.coli strain CD18 was cloned, sequenced and expressed in E.coli.