感染过程中钩端螺旋体外膜蛋白抗原表达水平变化及其调控机制  

Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism

作　　者：郑林立[1] 葛玉梅[1] 胡玮琳[1] 严杰[1] 

机构地区：[1]浙江大学医学院病原生物学系,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2013年第2期156-163,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(81261160321;81171534)

摘　　要：目的:了解感染人巨噬细胞过程中问号钩端螺旋体(简称钩体)黄疸出血群赖型赖株主要外膜蛋白(OMP)抗原表达水平变化及其OmpR相关基因调控机制。方法:采用生物信息学技术预测问号钩体赖株OmpR及其组氨酸激酶(HK)基因和结构功能域。采用实时荧光定量RT-PCR检测钩体感染人THP-1巨噬细胞前后钩体主要OMP编码基因mRNA水平的变化。采用HK胞外区多肽抗血清封闭试验及氯氰碘柳胺阻断试验,检测OmpR及其HK对感染过程中问号钩体OMP编码基因mRNA水平变化的影响。结果:生物信息学分析结果显示,LB015和LB333可能是问号钩体赖株OmpR编码基因,LB014可能是其HK编码基因。问号钩体赖株感染THP-1细胞后,lipL21、lipL32、lipL41基因mRNA水平迅速并持续下降(P<0.01),groEL、mce、loa22、ligB基因mRNA水平瞬时迅速升高(P<0.01)。氯氰碘柳胺或HK抗血清处理可使感染THP-1细胞而显著下降的问号钩体lipL21和lipL48基因mRNA水平明显回升(P<0.01),氯氰碘柳胺处理可使感染THP-1细胞而显著升高的groEL、mce、loa22、ligB基因mRNA水平明显下降(P<0.01)。结论:问号钩体赖株感染人巨噬细胞后主要OMP抗原表达水平发生明显变化。问号钩体赖株染色体中存在一组编码OmpR及其HK的基因,其主要功能可能是下调感染过程中部分OMP抗原表达水平。Objective： To determine expression changes of major outer membrane protein （OMP） antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. Methods： OmpR encoding genes and OmpR-re｜ated histidine kinase （HK） encoding gene of L. interrogans strain Lai and their functional domains were predicted using bioinformatics technique, mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT- PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. Results： The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated（ P 〈 0.01 ） , whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated（ P 〈 0.01 ）. The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes（P 〈 0.01 ）. Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes （ P 〈 0.01 ）. Conclusions ： Expression levels of L. interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR- and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

关 键 词：问号钩端螺旋体 细菌外膜蛋白质类 基因表达 逆转录聚合酶链反应 感染 外膜蛋白 表达 OMPR 氯氰碘柳胺 

分 类 号：R392.1[医药卫生—免疫学]