问号钩端螺旋体ompA基因分析及其重组表达产物的免疫学鉴定  

Analysis of Leptospira interrogans ompA gene and immunological identification of its recombinant expression product

作　　者：丁威[1] 董海艳[2] 薛峰[2] 严杰[2] 楼永良[1] 

机构地区：[1]温州医学院检验医学院微生态学研究所,325035 [2]浙江大学医学院病原生物学系

出　　处：《中华微生物学和免疫学杂志》2009年第4期370-374,共5页Chinese Journal of Microbiology and Immunology

基　　金：传染病国家科技重大专项项目（2008ZX10404）;浙江省科技厅国际合作重点项目（2006C24003）

摘　　要：目的了解我国15群15株问号钩端螺旋体（简称问号钩体）参考标准株携带ompA基因情况，重组表达OmpA（rOmpA）并鉴定rOmpA的免疫原性和免疫保护性。方法采用酚．氯仿法提取问号钩体基因组DNA，PCR扩增全长ompA基因，T—A克隆后测序。构建问号钩体黄疸出血群赖型56601株ompA基因的原核表达系统，采用SDS—PAGE及Bio—Rad凝胶图像分析系统检测rOmpA表达情况及其产量。rOmpA免疫家兔以获得抗血清，采用免疫扩散试验检测抗血清效价。采用Western blot检测rOmpA与其抗血清和问号钩体56601株全菌抗血清的免疫反应性，显微镜凝集试验（MAT）检测rOmpA抗血清对15株问号钩体的交叉凝集情况。分别采用问号钩体黏附J774A．1细胞模型和豚鼠感染模型，了解rOmpA兔抗血清黏附阻断及rOmpA免疫保护作用。结果15株问号钩体均含有序列保守的ompA基因，双曲钩体Patoc Ⅰ株则否。rOmpA表达量约占细菌总蛋白的20％。rOmpA能诱导家兔产生抗体，其抗血清免疫扩散效价为1：4。兔抗血清及问号钩体56601株全菌抗血清均能与rOmpA产生阳性Western blot信号。rOmpA抗血清对15株问号钩体的MAT效价为1：20～1：320。1：10～1：160稀释的rOmpA抗血清均能阻断问号钩体黏附J774A．1细胞，100μg和200μgrOmpA对豚鼠的免疫保护率分别为50．0％和75．0％。结论ompA基因仅存在于不同血清群致病性问号钩体基因组中。rOmpA具有较好的抗原性，多种免疫学方法检测显示，有可能作为通用型问号钩体基因工程疫苗的候选抗原。Objective To investigate the distribution of ompA gene in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogans, and to express recombinant OmpA （rOmpA） and to identify immunogenicity and immunoprotection of rOmpA. Methods Genomie DNAs from different leptospiral strains were extracted by phenol-chloroform method. Entire ompA gene fragments from the strains were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of ompA gene from L. interrogans strain 56601 was constructed, and the expression and yield of rOmpA were determined by SDS-PAGE plus Bio-Rad Agarose Image Analyser. Rabbits were immunized with rOmpA for obtaining antiserum, and immunodiffusion test was used to measure the antiserum＇s titer. Western blot assay was performed to determine the immunoreactivity of rOmpA with the antiserum against rOmpA and antiserum against whole cell of L. interrogans strain 56601, while microscopic agglutination test （MAT） was applied to detect the cross agglutination to the 15 L. interrogans strains. A leptospire adhering cell model and a leptospire infecting guinea pigs model were used to determine the adhesion-blocking effect of rOmpA antiserum and immunoprotection of rOmpA. Results All the 15 L. interrogans strains, but not L. biflexa strain Patoc Ⅰ , had sequence conserved ompA genes. The yield of rOmpA was approximate 20% of the total bacterial proteins, rOmpA could induce rabbits to produce antibody and immunodiffusion titer of the antiserum was 1：4. Both antisera against rOmpA and against whole cell of L. interrogans strain 56601 were able to produce positive Western blot signs to rOmpA, and the former offered 1：20-1：320 MAT titers to the 15 L. interrogans strains. 1： 10-1：160 dilutions of rOmpA antiserum could efficiently block L. interrogans strain 56601 adhering to J774A. 1 cells, and 100μg and 200μg rOmpA displayed 50.0% and 75.0% immunoprotective rates in the infected guinea pigs. Conclusion ompA gene only exists in

关 键 词：问号钩端螺旋体 ompA基因 重组表达 免疫原性 免疫保护性 

分 类 号：R392[医药卫生—免疫学]