问号钩端螺旋体血清群LipL41基因型分析及其表达产物的免疫学鉴定

Genotyping of LipL41 genes from Leptospira interrogans serogroups and immunological identification of the expression products

目的 确定我国 15群 15株问号钩端螺旋体 (简称钩体 )参考标准株和 2群 2株双曲钩体国际标准株携带LipL4 1基因情况 ,构建该基因的原核表达系统 ,鉴定表达产物的免疫原性。方法常规酚 氯仿法提取上述 17株钩体基因组DNA ,高保真PCR扩增全长LipL4 1基因片段 ,T A克隆后测序分型。构建LipL4 1基因原核表达系统 ,SDS PAGE检测重组目的蛋白 (rLipL4 1)表达情况。分别用钩体属特异性TR patocⅠ抗原、rLipL4 1兔抗血清的Westernblot鉴定其免疫反应性和抗原性。分别用显微镜凝集试验 (MAT)、钩体黏附J774A .1细胞模型检测兔抗rLipL4 1血清的交叉凝集效价和黏附阻断作用。结果 15株问号钩体均有LipL4 1基因 ,并可分为LipL4 1 1和LipL4 1 2两种基因型 ,2株双曲钩体则否。 11个LipL4 1 1基因和 4个LipL4 1 2基因克隆之间的核苷酸和氨基酸序列相似性分别为88.6 1%～ 88.6 7%和 93.2 4 %～ 97.18%。所构建的原核表达系统rLipL4 1 1和rLipL4 1 2的表达量分别占钩体总蛋白的 30 %和 4 0 %。rLipL4 1 1和rLipL4 1 2均能与TR patocⅠ抗血清发生结合反应 ,免疫家兔能产生抗体。rLipL4 1 1和rLipL4 1 2兔抗血清对上述 15株问号钩体MAT效价为 1∶8～ 1∶12 8、1∶16～1∶2 5 6稀释时均能有效地阻断钩体对细胞的黏附。结?

Objective To determine the existence of LipL41 gene in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogans and 2 international standard strains belonging to 2 serogroups of Leptospira biflexa for understanding physical expression of the gene in the strains, construct prokaryotic expression system of the gene and identify immunity of the expression products. Methods Genomic DNAs from the 17 strains of Leptospira were prepared by routine phenol-chloroform method. Entire LipL41 gene fragments from the strains were amplified by high fidelity PCR. The target amplification products were sequenced for genotyping after T-A cloning and then prokaryotic expression systems of the cloned LipL41 genes were constructed. By using SDS-PAGE, expression of the recombinant proteins (rLipL41) was identified. Western blot assays using rabbit antiserum against genus-specific leptospiral TR/patocⅠ antigen and rabbit anti-rLipL41 sera were applied to determine immunoreactivity and immunogenicity of rLipL41 respectively. Microscopic agglutination test (MAT) was performed to examine across agglutination titers of the rabbit anti-rLipL41 sera and J774A.1 cell adherence model of Leptospira was used to determine blocking effects of anti-rLipL41 sera. Results All strains but 2 of Leptospira biflexa genes, which can be divided into two genotypes LipL41/1 and LipL41/2. Similarities of the nucleotide and putative amino acid sequences of LipL41/1 genotypes from 11 of the strains and LipL41/2 genotypes from 4 of the strains were 88.61%-88.67% and 93.24%-97.18% respectively. Outputs of the rLipL41/1 and rLipL41/2 expressed by the constructed prokaryotic expression systems were 30% and 40% of the total bacterial proteins respectively. Both the rLipL41/1 and rLipL41/2 were able to combine to the TR/patocⅠ antiserum and induce rabbits to produce antibodies. MAT titers of the rabbit anti-rLipL41/1 and anti-rLipL41/2 sera with the 15 strains of Leptospira interrogans were 1∶8-1∶128 and 1∶16-1∶256 dilutions of the antisera could efficiently block adherence of Leptospira to the cells. Conclusion The representatives of 15 Chinese dominant Leptospira interrogans serogroups possessed LipL41/1 or LipL41/2 gene. The prokaryotic expression systems constructed in this study could efficientlyexpressrLipL4 1 1andrLipL4 1 2 ,respectively .rLipL4 1 1andrLipL4 1 2weredemonstratedasthesurfaceprotein antigenswithwellimmunogenicityandantigenicityandwidelyexistedinthedifferentserogroupsofLeptospirainter rogans.