摘要
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。
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.
出处
《热带医学杂志》
CAS
2006年第5期518-520,506,共4页
Journal of Tropical Medicine
基金
军队"十五"指令性课题专题(No.01L050)
军队重点实验室专项基金
