摘要
目的 :构建 lt B/ ct B- omp L1/ 1融合基因及其原核表达系统 ,鉴定表达产物的免疫和佐剂活性 ,检测问号钩端螺旋体 (简称钩体 )野生株 omp L 1基因的携带和表达情况及钩体患者血清特异性抗体水平。方法 :采用连接引物 PCR构建 lt B- omp L 1/ 1和 ct B- omp L 1/ 1融合基因 ,常规方法构建其原核表达系统。采用 SDS- PAGE、Westernblot和 GM1- ELISA分别检测目的重组蛋白 r LTB- r Omp L 1/ 1和 r CTB- r Omp L 1/ 1表达量、免疫反应性及与 GM1结合的活性。采用 PCR和 MAT分别检测 97株问号钩体野生株 omp L1基因及其表达情况。采用 ELISA检测 2 2 8例钩体患者血清 omp L1基因产物的抗体。结果 :与报道的相关序列比较 ,lt B- omp L1/ 1和 ct B- omp L1/ 1融合基因核苷酸和氨基酸序列相似性 ,分别为 99.7%～ 99.9%和 99.5 %～ 10 0 %。r LTB- r Omp L1/ 1和 r CTB- r Omp L1/ 1表达产量均约为细菌总蛋白的 10 % ,主要以包涵体形式存在。 r LTB- r Omp L 1/ 1和 r CTB- r Omp L1/ 1均分别能与r Omp L 1/ 1兔抗血清和牛 GM1结合。 89.7%问号钩体野生株含有 omp L1基因 ,87.6 %问号钩体野生株分别与r Omp L 1/ 1和 r Omp L1/ 2兔抗血清出现效价 ,为 1∶ 4～ 1∶ 2 5 6的 MAT阳性结果。 86 .8%和 88.6 %的患?
Objective: To construct prokaryotic expression systems of ltB/ctB ompL1 /1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products. Methods: The fusion genes ltB ompL1 /1 and ctB ompL1 /1 were constructed using linking primer PCR method.SDS PAGE was used to examine expression of the target recombinant proteins rLTB rOmpL1/1 and rCTB rOmpL1/1.Western blot and GM1 ELISA were used to measure the immunogenic and GM 1 binding activities of rLTB rOmpL1/1 and rCTB rOmpL1/1,respectively.PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains.Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method. Results: WTBZ The homogeneity of nucleotide and putative amino acid sequence of ltB ompL1 /1 and ctB ompL1 /1 fusion genes were 99.7%～99.9% and 99.5%～100%,in comparison with the reported corresponding sequences.Expression outputs of both rLTB rOmpL1/1 and rCTB rOmpL1/1,mainly present in inclusion body,accounted for 10% of the total bacterial protein.Both rLTB rOmpL1/1 and rCTB rOmpL1/1 could combine to rabbit anti rOmpL1/1 serum and bovine GM1. 89 7% of L.interrogans wild strains had ompL1 gene.87.6% of the wild L.interrogans strains presented positive results for MAT (titers:1∶4~1∶256) with the rabbit anti rOmpL1/1 or anti rOmpL1/2 sera.86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies,respectively. Conclusion: The fusion proteins,rLTB rOmpL1/1 and rCTB rOmpL1/1,showed high immunogenic and GM 1 binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross antigenicity.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2005年第1期21-26,共6页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金项目 (39970 6 78)
