问号钩端螺旋体rLTB/rCTB-LipL32/1免疫保护作用及野生株LipL32基因表达和患者抗体检测  

Immunoprotection of Leptospira interrogans rLTB/rCTB-LipL32/1 and detection of LipL32 gene expression in the wild strains and antibody in sera of the patients

作　　者：阮萍[1] 严杰[2] 毛亚飞[2] 周晓辉[2] 

机构地区：[1]浙江绍兴文理学院医学院病原生物学教研室,绍兴312000 [2]浙江大学医学院病原生物学教研室,杭州310031

出　　处：《中华微生物学和免疫学杂志》2005年第5期369-374,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　构建LTB- LipL32/ 1和CTB- LipL32 / 1融合基因原核表达系统并鉴定其表达产物的免疫和佐剂活性及保护作用,了解问号钩端螺旋体(简称钩体)野生株LipL32基因携带、表达及钩体病人血清LipL32基因产物抗体产生频率。方法　采用常规分子生物学技术构建LTB- LipL32 / 1和CTB- LipL32 /1融合基因及其原核表达系统。采用SDS PAGE检测目的重组蛋白rLTB -LipL32 / 1及rCTB -LipL32 / 1表达情况。分别采用Westernblot和GM1 -ELISA检测rLTB -LipL32 / 1及rCTB -LipL32 /1的免疫反应性和佐剂活性。采用PCR和MAT分别检测97株问号钩体野生株LipL32基因及其表达情况。采用ELISA检测2 2 8例钩体病人血清LipL32基因产物的抗体。采用豚鼠保护试验检测重组蛋白的免疫保护作用。结果　与报道的相关序列比较,LTB -LipL32 /1和CTB- LipL32 / 1融合基因核苷酸序列相似性分别为99.12 %～99.71%和98.5 4 %～99.4 2 % ,氨基酸序列相似性分别为97.5 8%～99.6 3%和96 .77%～99.6 3%。rLTB- LipL32 1和rCTB- LipL32 /1表达产量均约为细菌总蛋白的10 % ,并主要以包涵体形式存在。rLTB -LipL32 / 1和rCTB- LipL32 /1均分别能与LipL32 /1兔抗血清和牛GM1结合。97.9%(95 97)问号钩体野生株含有LipL32基因,95 .9% (93 97)问?Objective To construct prokaryotic expression sy stems of LTB-LipL32/1 and CTB-LipL32/1 fusion genes and identi fy immune and adjuvant activities and protective effect of the products, and to understand frequencies of LipL32 gene carrying and expression in L.interro gans wild strains and antibody against product of the gene in sera from leptos pirosis patients. Methods By using routine molecular biol ogical techniques, LTB-LipL32/1 and CTB-LipL32/1 fusion genes and their prokaryotic expression systems were constructed. SDS-PAGE was applied to examine expression of the target recombinant proteins rLTB-LipL32/1 and rCT B-LipL32/1. By using Western blot and GM1-ELISA, immunoreactivity and adjuvant activity of rLTB-LipL32/1 and rCTB-LipL32/1 were measured, respectively. PCR was performed to detect LipL32 gene and MAT was used to determine expression of the gene in 97 wild strains of L.interrogans. Antibody against product o f LipL32 gene in serum samples from 228 leptospirosis patients by ELISA. Gui nea pig protective assay was used to measure immunoprotection of the recombi- nant proteins. Results In comparison with the reported co rresponding sequences, nucleotide sequence similarities of LTB-LipL32/1 and CTB-LipL32/1 fusion genes were 99.12%-99.71% and 98.54%-99.42%, resp ectively, while putative amino acid sequence similarities of the two genes were 97.58%-99.63% and 96.77%-99.63%, respectively. Expression outputs of both the rLTB-LipL32/1 and rCTB-LipL32/1, mainly present with inclusion bodies, were ap proximate 10% of the total bacterial proteins. rLTB-LipL32/1 and rCTB-LipL32/1 could combine rabbit anti-LipL32/1 serum as well as bovine GM1, respectively. 97.9% of the L.interrogans wild strains (95/97) had LipL32 gene and MAT results of 95.9% of the L.interrogans wild strains (93/97) and rabbit anti- rLipL32/1 or anti-rLipL32/2 serum were positive. 97.4% (222/228) and 94.7% (21 6/228) of the patients′ serum samples were positive for rLipL32/1 and rLipL32/2 antibodies, respectively. The immun

关 键 词：问号钩端螺旋体 lipL32基因 大肠埃希菌 LTB基因 霍乱弧菌 CTB基因 免疫性佐剂活性 

分 类 号：R392[医药卫生—免疫学] R446.6[医药卫生—基础医学]