| 阮萍,严杰,毛亚飞,彭慧琴,周晓辉.问号钩端螺旋体lipL41基因表达及重组抗原分析[J].Chinese journal of Epidemiology,2005,26(8):608-612 |
| 问号钩端螺旋体lipL41基因表达及重组抗原分析 |
| Identification on the immunogenic activity of recombinant rLTB/CTB-LipL41/ 1 to Leptospira interrogans and detection of lipL41 gene with its production |
| Received:October 21, 2004 |
| DOI： |
| KeyWord: 问号钩端螺旋体 融合基因 分析 |
| English Key Word: Leptospira interrogans Fusion gene Analysis |
| FundProject:国家自然科学基金资助项目(39970678) |
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| Abstract: |
| 目的构建问号钩端螺旋体(钩体)ltB/ctBlipL41/1融合基因及其原核表达系统。方法采用连接引物聚合酶链反应(PCR)构建ltBlipL41/1和ctBlipL41/1融合基因,常规方法构建其原核表达系统。采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳检测目的重组蛋白rLTBrLipL41/1和rCTBrLipL41/1表达情况;用免疫印迹和神经节苷脂酶联免疫吸附试验(GM1ELISA)分别检测上述目的重组蛋白的免疫原性和佐剂活性;采用PCR和显微镜凝集试验(MAT)分别检测97株问号钩体野生株lipL41/1基因及其表达情况;用ELISA检测228例钩体患者血清lipL41基因产物的抗体。结果与报道的相关序列比较,ltBlipL41/1和ctBlipL41/1融合基因核苷酸和氨基酸序列相似性分别为99.6%～99.9%和99.8%～100%。rLTBrLipL41/1和rCTBrLipL41/1表达产量均约为细菌总蛋白的10%,主要以包涵体形式存在。rLTBrLipL41/1和rCTBrLipL41/1均分别能与rLipL41/1兔抗血清和牛GM1结合。87.6%(85/97)问号钩体野生株含有lipL41基因,84.5%(82/97)问号钩体野生株分别与rLipL41/1和rLipL41/2兔抗血清出现效价范围为1∶4～1∶128的MAT阳性结果。84.6%(193/228)和78.5%(179/228)的患者血清分别rLipL41/1和rLipL41/2抗体阳性。结论成功地构建了ltBlipL41/1和ctBlipL41/1融合基因及其原核表达系统。所表达的rLTBrLipL41/1和rCTBrLipL41/1融合蛋白有良好的免疫原性和佐剂活性。lipL41基因存在于不同问号钩体血清群中并高频率表达。rLTBrLipL41/1和rCTBrLipL41/1具有成为钩体属特异性疫苗抗原的良好前景。 |
| English Abstract: |
| Objective To construct prokaryotic expression systems of ltB/ctB-lipL41/1 fusion genes, identify immunogenic and adjuvant activities of the products as well as to understand the frequencies of lipL41 gene that carrying and expressing in L.interroganswild strains and specific antibody levels in sera from patients with leptospirosis. Methods Polymerase chain reaction(PCR) with linking primer was applied to construct the fusion genes ltB-lipL41/1 and ctB-lipL41/1. By routine molecular biological techniques, prokaryotic expression systems of the two fusion genes were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was used to examine expression of the target recombinant proteins rLTB-rLipL41/1 and rCTB-rLipL41/1. Both western blot and Ganglioside-enzyme linked immunosorbent assay(GM1-ELISA) were used while immunogenic and adjuvant activities of rLTB-rLipL41/1 and rCTB-rLipL41/1 were measured. PCR and MAT were performed to detect lipL41 gene and expression of the gene in 97 wild strains of L.interrogans, respectively. Antibodies against product of lipL41 gene in serum samples from 228 leptospirosis patients were detected by ELISA. Results In comparisonwith reported corresponding sequences, the similarities of ltB-lipL41/1 and ctB-lipL41/1 fusion genes to nucleotide and putative amino acid sequence were 99.6%- 99.9%and 99.8%-100%, respectively. Expression outputs of both rLTB-rLipL41/1 and rCTB-rLipL41/1, mainly presenting with inclusion body, consisting approximate 10% of the total bacterial proteins. Both rLTB-rLipL41/1 andrCTB-rLipL41/1 could combine rabbit anti-rLipL41/1 serum as well as bovine GM1, respectively. 87.6% of the L.interrogans wild strains(85/97) having lipL41 gene while 84.5%(82/97) of the wild strains with rLipL41/1 or rLipL41/2 antiserum were positive for MAT with titers of 1∶4- 1∶128. 84.6%(193/228), 78.5%(179/228) from the patients' serum samples were positive for rLipL41/1 and rLipL41/2 antibodies, respectively. Conclusion ltB-lipL41/1 and ctB-lipL41/1 fusion genes and their prokaryotic expression systems were successfully constructed in this study. The two expressed fusion proteins showed qualified immunogenic and adjuvant activities. lipL41 gene was extensively distributed and frequently expressed in different serogroups of L.interrogans. rLTB-rLipL41/1 or rCTB-rLipL41/1 seemed to have had good potential to serve as an antigen in L.interrogans genus-specific vaccine. |
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