问号钩端螺旋体属特异性外膜蛋白LipL41抗原表位预测及免疫学鉴定  

Prediction and immunologic identification of antigenic epitopes in genus-specific outer membrane protein LipL41 of Leptospira interrogans

作　　者：姜久昆[1] 林旭瑷[1] 严杰[1,2] 薛峰[1] 

机构地区：[1]浙江大学医学院病原生物学系,浙江杭州310058 [2]浙江大学附属第一医院传染病诊治国家重点实验室基础医学微生物学研究室,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2008年第6期585-591,621,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(30671863);国家博士后基金资助项目(20060401070)

摘　　要：目的:预测及筛选问号钩端螺旋体(简称钩体)属特异性外膜蛋白LipL41有效T和B细胞(T/B)联合表位,了解不同基因型LipL41s差异T/B联合表位的免疫反应性差别。方法:采用生物信息学方法预测LipL41/1和LipL41/2分子中T/B联合表位。采用PCR扩增候选T/B联合表位片段,采用噬菌体展示及SDS-PAGE等技术获得含不同T/B联合表位的重组P。分别以rLipL41/1和rLipL41/2抗血清、黄疸出血群赖型赖株全菌抗血清和钩体患者血清为一抗,采用Western Blot检测各抗血清与各重组P免疫杂交反应性。结果:根据预测结果选择了LipL41s中8个共有或差异T/B联合表位。经PCR扩增获得了上述T/B联合表位片段。各T/B联合表位片段均准确插入噬菌体P蛋白N端并有效表达。各抗血清均能识别上述8个T/B联合表位,但其WesternBlot杂交信号强度存在差异,其中共有T/B联合表位LipL41/1-30和LipL41/1-233与不同抗血清的信号较强且稳定。结论:所选择的8个T/B联合表位均为LipL41的有效抗原表位。共有T/B联合表位LipL41/1-30和LipL41/1-233可作为钩体MAP疫苗的首选抗原表位。差异T/B联合表位LipL41s-89和LipL41s-299与不同抗血清有免疫交叉现象。Objective： To predict and screen the efficient antigenic epitopes in genus-specific envelope protein LipL41 of Leptospira interrogans and to determine the immunoreactive diversity of LipL41s from different genotypes. Methods. Bioinformatic methods were applied to predict the T/B combined epitope candidates in LipL41/1 and LipL41/2 molecules. The nucleotide fragments encoding epitopes were amplified by PCR. Phage display system with SDS-PAGE was performed to obtain the recombinant PⅢs containing different T/B combined epitopes. Western Blot assays were performed to determine the immunoreactivity of recombinant PⅢs to various antisera including antiserum against rLipL41/1 ,rLipL41/2 and whole cell of L. interrogans strain Lai,and serum from patients with leptospirosis. Results. Based on the predicting data,eight common or differential combined epitopes in LipL41s were selected. The nucleotide fragments encoding the epitopes were obtained by PCR. All the T/B combined epitope fragments were correctly inserted into the N end of phage PⅢ protein and then successfully expressed. All the antisera were able to recognize each of the epitopes but the hybridization signal intensity was different. Among these epitopes,the common T/B combined epitopes LipL41/1-30 and LipL41/1-233 showed a stronger and stable hybridization signals. Conclusion. All 8 selected T/B combined epitopes in the study are the efficient antigenic epitopes. The common T/B epitopes LipL41/1-30 and LipL41/1-233 can be first used in development of leptospiral MAP vaccine. The cross immunoreaction is between the differential T/B epitopes LipL41s 89,LipL41s-299 and the different antisera.

关 键 词：钩端螺旋体 问号/免疫学 细菌外膜蛋白质类/生物合成 细菌外膜蛋白质类/分离 外膜脂蛋白/属特异性抗原 LipL41/1/LipL41/2 抗原表位/预测 噬菌体展示 免疫学鉴定 

分 类 号：R392[医药卫生—免疫学]