不同血清群问号钩端螺旋体mce基因序列分析及表达模式的研究

Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups

目的:确定问号钩端螺旋体(简称钩体)株携带侵袭相关mce基因情况,原核表达mce基因并制备其抗血清,了解问号钩体感染细胞前后mce基因转录水平的变化。方法:采用PCR扩增13株问号钩体和1株双曲钩体全长mce基因片段,T-A克隆后测序。采用基因工程技术构建mce基因原核表达系统,SDS-PAGE联合Bio-Rad凝胶图象分析系统检查目的重组蛋白rMce表达情况,采用Western Blot对表达的rMce进行鉴定。采用皮内免疫法制备兔抗rMce血清,免疫双扩散试验测定其效价。采用实时荧光定量RT-PCR,检测问号钩体黄疸出血群赖型56601株感染J774A.1细胞前后mce基因转录水平的变化。结果:我国13株问号钩体株均携带mce基因,双曲钩体三宝垄群patoc型Patoc株则否。与报道的序列(GenBank accession No.:NP_712236)比较,所克隆的mce基因核苷酸和氨基酸序列相似性分别为99.02%～100%和97.91%～100%。所构建的mce基因原核表达系统能表达rMce,其产量为细菌总蛋白的5%。问号钩体56601株全菌抗血清能有效识别rMce。问号钩体56601株感染细胞后mce基因转录水平明显上调。结论:mce基因仅存在于致病性的问号钩体中,且其转录水平呈细胞接触上调模式,表明该基因与问号钩体致病性密切相关。mce基因产物是序列保守的外膜蛋白。所构建的mce基因原核表达系统及所制备的rMce抗血清为深入研究该基因功能提供了有效的手段。

Objective： To determine the frequency of race gene in Leptospira interrogans,and to investigate the gene transcription levels of L. interrogans before and after infecting cells. Methods： The segments of entire race genes from 13 L. interrogans strains and 1 L. biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of race gene was constructed ; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum,the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A. 1 cells were monitored by real-time fluorescence quantitative RT-PCR. Results ： race gene was carried in all tested L. interrogans strains,but not in L. biflexa serogroup Semaranga serovar patoc strain Patoc I . The similarities of nucleotide and putative amino acid sequences of the cloned rnce genes to the reported sequences （GenBank accession No：NP 712236） were 99. 02%- 100% and 97. 91%- 100%,respectively. The constructed prokar;otic expression system of race gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L. interrogans strain 56601 efficiently recognized rMce. After infecting J774A. 1 cells,transcription levels of the race gene in L. interrogans strain 56601 were remarkably up-regulated. Conclusion： The constructed prokaryotie expression system of rnce gene and the prepared antiserum against rMee provide useful tools for further study of the gene function.