【摘要】 目的　探讨 15群 15型问号钩端螺旋体 (简称钩体 )中国参考标准株及 2群 2型双曲钩体国际参考标准株是否均存在主要外膜蛋白 (MOMP)基因 (LipL32 ) ,克隆并构建该基因的原核表达系统 ,鉴定表达产物的免疫性。方法　常规酚 氯仿法提取上述钩体株基因组DNA ,高保真PCR扩增全长LipL32基因片段 ,T A克隆后测定核苷酸序列并构建表达系统 ,不同浓度IPTG诱导后用SDS PAGE检测rMOMP表达情况。分别用兔抗TR patocⅠ钩体全菌抗血清、rMOMPs免疫兔血清的Westernblot鉴定其免疫反应性和免疫原性 ,显微镜凝集试验 (MAT)检测rMOMPs免疫兔血清的交叉凝集效价 ,钩体细胞黏附模型检测抗体阻断效果。结果　上述 17株钩体均有LipL32基因 ,但可分LipL32 1和LipL32 2两种基因型。 13个LipL32 1和 4个Li pL32 2基因型之间核苷酸和氨基酸序列同源性分别为 95 .12 %～ 96 .6 0 %和 97.79%～ 98.16 %。IPTG诱导后rMOMP1和rMOMP2表达量分别占细菌总蛋白的 4 0 %和 10 %。rMOMP1和rMOMP2均能与兔抗钩体TR patocⅠ血清发生结合反应 ,免疫家兔可对上述 17株钩体产生 1∶2～ 1∶6 4MAT效价的凝集抗体。 1∶2～ 1∶16稀释的兔抗rMOMP1和rMOMP2血清均能有效地阻断钩体的黏附。结论　所有检测的钩体具有LipL32 1或LipL32 2基因。所更多还原

【Abstract】 Objective To determine the existence of major outer membrane protein (MOMP) gene (LipL32) in 15 Chinese dominant strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression systems of the gene, and to identify immunity of the expression products. Methods Genomic DNAs from the strains of leptospira were prepared by using routine phenol-chloroform method. The fragments of LipL32 gene of whole length were amplified by high fidelity PCR. The target amplification products were sequenced after T-A cloning and the expression systems of the gene were constructed. Expression of the recombinant proteins were identified by using SDS-PAGE after induction with IPTG of different dosages. Western blot assays were applied to determine immunoreactivity and immunogenicity of the recombinant proteins. Cell adherence model of leptospira was used to examine the blocking effects of rabbit antisera against the two rMOMPs. Results All the tested strains of leptospira had LipL32 gene which could be divided into two genotypes LipL32/1 and LipL32/2. Homologies of the nucleotide and putative amino acid sequences of 13 LipL32/1 gene and 4 LipL32/2 gene fragments were 95.12%-96.60% and 97.79%-98.16% respectively. Outputs of rMOMP1 and rMOMP2 induced by IPTG were 40% and 10% of the total bacterial proteins, respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/patocⅠ. rMOMP1 and rMOMP2 induced agglutination antibodies with 1∶2-1∶64 MAT tiers for the 17 strains of leptospira. The rabbit anti-rMOMP1 and anti-rMOMP2 sera at 1∶2-1∶16 dilutions could efficiently block adherence of leptospira. Conclusion All the leptospira tested possessed LipL32/1 or LipL32/2 gene. The constructed expression systems could express rMOMP1 and rMOMP2, respectively. rMOMP1 and rMOMP2 showed satisfied immunogenicity and immunoreactivity. rMOMP1 and rMOMP2 should be the genus-specific protein antigens located on the surface of leptospira and could induce cross agglutination and adherence blocking antibodies.更多还原