中国主要问号钩端螺旋体血清群外膜蛋白OmpL1的基因型,原核表达系统构建表达及免疫学鉴定  

Genotypes of the OmpL1 gene from the dominant serogroups of Leptospira interrogans in China and construction of prokaryotic expression system of the gene and immunological identification of the recombinant protein

作　　者：徐飏[1] 严杰[2] 毛亚飞[2] 李立伟[2] 李淑萍[2] 

机构地区：[1]华中科技大学同济医学院病原生物学系 [2]浙江大学医学院病原生物学教研室

出　　处：《中华微生物学和免疫学杂志》2004年第6期439-444,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 3 9970 678)

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外膜蛋白OmpL1基因 ,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的基因组DNA ,高保真PCR扩增全长OmpL1基因片段 ,T A克隆后测定核苷酸序列并构建原核表达系统 ,不同浓度IPTG诱导后用SDS PAGE检测重组蛋白rOMPL1表达情况。分别用兔抗钩体TR PatocⅠ抗原、rOMPL1血清的Westernblot鉴定其免疫反应性和免疫原性。分别用显微镜凝集试验 (MAT)和钩体细胞黏附模型检测兔抗rOMPL1血清的交叉凝集效价和细胞黏附阻断作用。结果　上述 17株钩体均具有OmpL1基因 ,但至少可分 3种基因型 :OmpL1 1、OmpL1 2和OmpL1 3。与文献报道比较 ,所克隆的OmpL1 1、OmpL1 2和OmpL1 3基因核苷酸序列同源性分别为 93.87%～ 94 .0 8%、89.82 %～ 10 0 %和 80 .89%～ 81.72 % ,其氨基酸序列同源性分别为 93.13%～ 98.75 %、91.87%～ 10 0 %和 85 .6 3%～ 88.4 4 %。IPTG诱导后pET32a OmpL1 1 E .coliBL2 1DE3和pET32a OmpL1 2 E .coliBL2 1DE3的rOMPL1 1和rOMPL1 2表达量分别占细菌总蛋白的 30 %和 2 0 %。rOMPL1 1和rOMPL1 2均能与兔抗钩体TR PatocⅠ抗原血清发生免疫结合?Objective To determine the existence of OmpL1 gene in 15 dominant Chinese strains from 15 serogroups of Leptospira interrogans and 2 international strains from 2 serogroups of Leptospira biflexa, to clone and construct the expression systems of the gene and to identify the immunogenicity of the expression products. Methods The target amplification products were sequenced after T-A cloning and prokaryotic expression systems of the gene were constructed. Expression of the recombinant proteins were demonstrated by SDS-PAGE. Western blot was applied to determine immunoreactivity and immunogenicity of the recombinant proteins. Results All the leptospiral strains tested had OmpL1 gene. These OmpL1 genes were divided into three genotypes: OmpL1/1, OmpL1/2 and OmpL1/3. In comparison with the reported sequences of OmpL1 gene, nucleotide sequence homologies of OmpL1/1, OmpL1/2 and OmpL1/3 genotypes were 93.87%-94.08%, 89.82%-100% and 80.89%- 81.72% , respectively. When compared with the reported putative amino acid sequences, homologies of the three genotypes were 93.13%-98.75%, 91.87%-100% and 85.63%-88.44%, respectively. Both the rOMPL1/1 and rOMPL1/2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ antigen and induce rabbits antibodies. MAT titers of the rabbit anti-rOMPL1/1 and anti-rOMPL1/2 sera with the 17 strains of leptospira were 1∶2 -1∶32 and the antisera with 1∶2-1∶16 dilutions could efficiently block leptospira to adhere J744A.1 cell. Conclusion All the pathogenic and non-pathogenic leptospira tested possess OmpL1 gene, which can be at least divided into three different genotypes. rOMPL1/1 and rOMPL1/2 expressed by the constructed prokaryotic expression systems showed immunoreactivity and immunogenicity. rOMPL1/1 and rOMPL1/2 should be the genus-specific protein antigens located on the surface of leptospira and can induce across agglutinating and adherence blocking antibodies.

关 键 词：中国 问号钩端螺旋体 血清群外膜蛋白OmpL1 基因型 原核表达 系统构建 基因表达 免疫学 交叉凝集 

分 类 号：R392[医药卫生—免疫学]