赖型钩端螺旋体OmpA膜蛋白Loa22基因的克隆分析及蛋白表达

目的分析赖型钩端螺旋体OmpA膜蛋白Loa22的结构特征,并对其进行克隆表达。方法分别以问号状赖型钩体017株、56601株及双曲状钩体PatocI株基因组为模板PCR扩增目的基因。构建Loa22基因与质粒pGEX-4T-1的重组原核表达质粒,克隆筛选并测序。利用Bioedit、Dnaman、PSIPRED、NCBI及SignaIP等对其结构进行分析。最后在大肠杆菌JM109中诱导表达目的蛋白。结果不同毒力赖型钩体均能扩增出约600bp的片段,而PatocI株则未能扩增出目的片段;PCR、双酶切及测序证实pGEX-Loa22构建成功:分析显示不同赖型钩体的Loa22的同源性很高,C末端都具有同OmpA一致的保守序列及肽聚糖相关基序,都有信号肽序列;表达产物经SDS-PAGE检测证明是目的蛋白。结论赖型钩体具有编码OmpA膜蛋白的Loa22基因,可能与赖型钩体毒力和免疫原性存在某种相关性。

Cloning and expression of the OmpA membrane protein Loa22 gene of Leptospira Lai

To clone and analyze the membrane protein Loa22 Gene of Leptospira(L.)Lai and its expression in E.coil.the Loa22 gene was amplified by PCR from genome of L.Lai 017 strain,56601 strain and L.bilexa PatocI strain.The amplified DNA fragment was then cloned into vector pGEX-4T-1.and DNA sequence was performed subsequnently.Later,the structure of Loa22 was analyzed by Bioedit,Dnaman,PSIPRED,NCBI and SignaIP etc.Loa22 was expressed in E.coil JM109 in the end.It was found that about 600bp fragment was amplified in various virulent L.interrogans serovar Lai.but not in L.bilexa patocI strain.The recombinant plasmid was construced triumphantly.There was a high homology of DNA squence of the Loa22 in various L.Lai.Loa22 had a C-terminal OmpA domain and a signal peptide.The fusion protein GST-Loa22 was successfully expressed in E.coilJM109.These results suggested that Loa22 gene is the conservative pathogenic mark in Leptospira,it may be associaled with virulence and immunogenicity infection with pathogenic Leptospira.