赖型钩端螺旋体601株OmpL1蛋白的表达、纯化及功能分析

目的对钩端螺旋体黄疸出血群赖株ompL1基因进行克隆、表达及产物的纯化。方法从我国钩端螺旋体黄疸出血群赖株基因组中扩增全长ompL1基因片段,克隆至pGEM-T载体,回收经EcoRⅠ HindⅢ双酶切产物,将其与表达载体pET32a连接,通过酶切图谱和序列分析法筛选出重组质粒。将含重组质粒的宿主菌诱导表达后,提取全菌总蛋白进行SDS-PAGE电泳分析。结果获得pET9L重组质粒,经诱导表达后得到分子量为31000的重组蛋白。测序得到的ompL1基因核苷酸序列与已发表ompL1基因序列(GenbankI61405)同源性为99.8%,所推导的氨基酸序列(GenbankLA3138)的同源性达100%。结论成功构建了ompL1基因的原核表达系统,为进一步制备以OmpL1为检测靶抗原的免疫层析试剂盒奠定基础。

Cloning and Expression of OmpL1 Gene of Leptospira interrogans Serogroup Icterohaemorrhagiae Strain Lai

Objective To clone and express the ompL1 gene of Leptospira interrogans serogroup icterohaemorrhagiae strain Lai,and to purify its products.Methods The full-length ompL1 gene from the genome of Leptospira interrogans serogroup icterohaemorrhagiae strain Lai was amplified,and then cloned into pGEM-T vector.The genes were then cloned into the prokaryotic expression plasmid pET32a,and recombinant plasmids were screened by enzyme-cut and sequence analysis.The host bacteria containing recombinant plasmid were induced with IPTG,and the recombinant protein was analyzed by SDS-PAGE.Results The homology of nucleotide and the deduced amino acid sequence of the cloned ompL1 gene were 99.8% and 100% similar to the previously reported ompL1 gene(Genbank I61405) and protein(Genbank LA3138),respectively.Conclusion OmpL1 was successfully expressed in the prokaryotic expression system.OmpL1 may be used as a target antigen for detecting Leptospira using immunological methods.