我国主要问号钩端螺旋体血清群lipL21基因序列及其产物免疫反应性分析

Sequencing and immunoreactive analysis of lipL21 genes with its products from dominant serogroups of Leptospira interrogans in China

目的分析我国15群15型问号钩端螺旋体(简称钩体)参考标准株外膜脂蛋白lipL21基因序列,构建该基因原核表达系统并鉴定表达产物的免疫原性,了解lipL21基因自然表达状况。方法高保真PCR扩增上述问号钩体株及双曲钩体Patoc型PatocⅠ株基因组DNA中全长lipL21基因片段,T-A克隆后测序并构建其原核表达系统。分别用钩体TR/PatocⅠ和rLipL21兔抗血清为一抗的Western blot鉴定目的重组蛋白rLipL21的免疫原性,显微镜凝集试验(MAT)检测rLipL21兔抗血清的交叉凝集效价。以盐变-去垢剂处理法提取钩体外膜蛋白,用SDS-PAGE和免疫印迹法检测上述钩体株lipL21基因自然表达情况。结果上述钩体株均存在序列高度保守的lipL21基因,其核苷酸和氨基酸序列相似性分别为98．75％～99．82％和99．46％～100％。rLipL21能与TR/PatocⅠ及rLipl21兔抗血清发生结合反应。rLipL21免疫家兔能产生抗体,该抗体对上述15株问号钩体MAT效价为1：16～1：128。问号钩体外膜标本中均可检出LipL21,双曲钩体则否。结论我国钩体群参考标准株均含有序列保守的lipL21基因并自然表达于外膜,双曲钩体PatocⅠ株虽含有lipL21基因但未表达。rLipL21具有良好的抗原性和免疫反应性,有可能作为新型钩体疫苗或检测试剂盒的候选属特异性表面抗原之一。

Objective To analyze the sequences of lipL21 genes encoding an outer envelope lipoprotein of the representative strains from 15 serovars belonging to 15 serogroups of Leptospira interrogans with identification of immunagenicity of the expression products, and to understand the natural expression of the gene. Methods The entire lipL21 genes from genomic DNAs of the L.interrogans strains and L. biflexa serovar Patoc strain Patoc I were amplified by high fidelity PCR. The target amplification fragments were cloned for sequencing using T-A cloning method and a prokaryotic expression system of the gene was then constructed. The immunogenicity of the target recombinant protein rLipL21 was identified by Western blot assay. Microscopic agglutination test （MAT） was used to titrate rabbit anti-rLipL21 serum. The outer envelope proteins（OEP） of the leptospiral strains were prepared by using salt-denature and detergent treatment. SDS-PAGE and immunoblotting assay were applied to demonstrate the natural expression of the lipL21 genes of the leptospiral strains. Results All the tested leptospiral strains had lipL21 genes with high conserved sequences and homology of their nucleotide and putative amino acid sequences were 98.75%-99.82% and 99.46%-100%, respectively, rLipL21 combined with both rabbit antisera against TR/Patoc I or rLipL21, rLipL21 was able to induce the immunized rabbits producing antibody response. The Li- pL21 expression in all the OEP preparations from L.interrogans strains was detectable but not in the OEP prepara- tion from L. biflexa strain Patnc I.Conclusion All the representative strains of L. tnterrogans serngroups dominant in China have sequence-ennserved lipL21 genes which naturally express their products in outer envelopes. L. biflexa strain Patoc I has a complete lipL21 gene sequence but no expression was found, rLipL21 showed fine antigenicity and immunoreactivity, indicating this protein has a powerful potential as a candidate of the leptospiral genus-specific surface antigens for developing a novel vaccine or a new detection kit of Leptospira.