问号钩端螺旋体铁离子调节蛋白A的免疫原性及保守性研究  

Research on immunogenicity and conservation of IRPA in Leptospira interrogans

作　　者：林玮[1] 杨宏亮[2] 胡宝瑜[2] 杨杨[2] 谭立志[1] 郭晓奎[2] 

机构地区：[1]南华大学病原生物学研究所,衡阳421001 [2]上海交通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》2007年第9期797-801,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(30670102;30370071);湖南省卫生厅科研基金(B2003-075)

摘　　要：目的克隆表达和鉴定问号钩端螺旋体(L.interrogans,简称钩体)黄疸出血群赖型赖株中铁离子调节蛋白A(IRPA),研究IRPA的免疫原性和在不同钩体菌种中的保守性,探讨其在致病和疫苗研究中的意义。方法生物信息学软件分析预测IRPA的特征。构建原核表达质粒pQE31- IRPA,经IPIG诱导后用SDS-PAGE及Western blot鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western blot检测其免疫原性和在不同血清型钩体中的保守性。ELISA和Western blot检测钩体全菌兔抗血清中的IRPA抗体。结果生物信息学预测结果显示,IRPA是可能位于外膜的脂蛋白,在钩体内有多个蛋白可能与之相互作用。成功克隆表达了重组质粒pQE31-IRPA,重组蛋白能刺激BALB/c小鼠产生抗体(效价为1:32 000),并能与相应抗体反应,具有良好的免疫原性。在15株不同血清型的问号钩体及1株双曲钩体(L.biflex)中均可检测到重组蛋白的表达,并在钩体全菌兔抗血清中检测到其抗体。结论IRPA具有良好的免疫原性和保守性,为进一步研究其在钩体病中的作用机制及其作为候选基因疫苗的研究奠定了基础。Objective To clone and express the iron-regulated protein A（IRPA） gene of Leptospira interrogans （ L. interrogans ） serogroup Icterohaemorrhagiae serovar Lai strain Lai, and to analyze the immunogenicity and conservation of the recombinant protein in different serovars of L, interrogans. Methods The character of IRPA was predicted by bioinformatics software, The prokaryotic expression system of the IRPA was constructed by routine techniques and expression of therecombinant proteins were identified by SDS-PAGE and Western blot after induction with IPTG. Western blot was used to identify the immunogenicity of IRPA and the conservation of expressed IRPA in sixteen different serovars, The antibody of IRPA in rabbit serum of anti-L, interrogans strain Lai was identified by Western blot and ELISA respectively. Results IRPA is an outer membrane lipoprotein in L, interrogans predicted by bioinformatics software. There are several proteins predicted interacting with IRPA, The prokaryotic expression system of IRPA was successfully constructed, The titer of anti-recombinant protein serum in BALB/c mouse was 1：32 000, The IRPA was detected in fifteen different serovars of L, interrogans and a L. biflexa, The rabbit serum of anti-L, interrogans strain Lai was positive for recombination IRPA antibody detected by Western blot and ELISA respectively. Conclusion IRPA of L, interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai shows expecting immunogenicity and conservation, Our studies on IRPA provide a basis for investigating the role of IRPA in pathogenesis of leptospirosis and development of vaccine.

关 键 词：问号钩端螺旋体 铁离子调节蛋白A 基因表达 免疫原性 保守性 

分 类 号：R377[医药卫生—病原生物学]