钩端螺旋体外膜蛋白LipL45相关基因的保守性分析及在诊断中的初步应用

Conservative analysis of gene encoding outer membrane lipoprotein LipL45 component and its potential value in the diagnosis

目的:分析外膜蛋白LipL45在不同致病性钩端螺旋体中的同源性,初步探讨其在钩端螺旋体病诊断中的应用价值。方法:克隆表达外膜蛋白LipL45相关基因,用酶联免疫吸附试验和显微凝集试验分别测定其抗血清效价和抗体活性。以15株不同致病性钩端螺旋体的基因组DNA为模板PCR扩增此外膜蛋白基因序列,通过测序进行同源性分析。表达产物作为包被抗原与部分钩端螺旋体患者血清反应,评估其作为诊断抗原的可行性。结果:免疫印迹法结果显示,抗血清能识别大多数致病性钩端螺旋体菌体蛋白,显微凝集试验结果也显示此外膜蛋白抗血清与大多数致病性钩端螺旋体凝集反应呈阳性。用重组LipL45蛋白包被96孔板作酶联免疫吸附试验检测显示,此蛋白抗原与22名感染者血清呈现不同水平的免疫阳性反应,而与10份正常人血清呈阴性反应(P<0.01)。核酸序列分析显示,LipL45相关基因在致病性钩端螺旋体L. interrogans中比较保守,在另2种致病性钩端螺旋体L. borgpetersenii和L.weilii中不存在或同源性较低。而在非致病性腐生型钩端螺旋体中未得到相应PCR扩增产物。结论:钩端螺旋体外膜蛋白LipL45相关基因作为问号钩端螺旋体的保守基因可用于钩端螺旋体的基因分型,其重组表达产物在钩端螺旋体诊断及疫苗研究中具有一定的应用前景。

Objective To investigate the conservative of gene encoding leptospiral outer membrane protein LipL45 among various leptospira strains and its potential value in the diagnosis of leptospirosis. Methods The gene encoding LipL45 from the genome sequence of L. interrogans serovar lai strain Lai was cloned and expressed. Purified proteins were identified by SDS-PAGE and ELISA. Nucleotide sequencing was adopted for the isogenous analysis of the related Lip 45 gene among various pathogenic leptospires. The purified protein product as coating antigen with sera from those infected by pathogenic leptospires was also analyzed. Results Both immunoblot analysis and MAT results revealed that the antisera to the LipL45 components reacted with the extracted proteins or whole cells of most involving pathogenic Leptospires. The positive reaction of the recombinant outer membrane protein with tested sera from 22 individual infected by leptospira and the negative results with sera from 10 normal individuals were detected （P〈0. 01）. Sequence analysis indicated that the LipL45 related gene was highly conserved among L. interrogans strains but absent or having poor homology in the strains of the other two pathogenic leptospira i. e. L. borgpetersenii and L. weilii. And the gene could not be amplified from the saprophytic L. biflexa strain. Conclusion The high conserved gene encoding LipL45 components among distinct pathogenic strains of L. interrogans may play a potential role in both genetic classification of leptospires and the diagnosis of leptospirosis as well as the research on vaccines.