【作者】 阮萍； 严杰； 毛亚飞； 李淑萍； 罗依惠； 李立伟；

【Author】 RUAN Ping~1 YAN Jie~2 MAO Ya-fei~2 et al1.Department of Medical Microbiology and Parasitology,Medical School of Shaoxing University,Shaoxing 312000;2.Department of Medical Microbiology and Parasitology,College of Medical Science,Zhejiang University,Hangzhou 310031,china

【机构】 绍兴文理学院医学院； 浙江大学医学院病原生物学教研室；

【摘要】 目的:构建ltB/ctB-ompL1/1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ompL1基因的携带、表达情况和钩体病人血清特异性抗体。方法:采用连接引物PCR 构建ltB-ompL1/1和ctB-ompL1/1融合基因,常规方法构建其原核表达系统。采用SDS-PAGE 检测目的重组蛋白rLTB-rOmpL1/1和rCTB-rOmpL1/1表达情况。采用Western Blot 和GM₁-ELISA 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MAT分别检测97株问号钩体野生株ompL1基因及其表达情况。采用ELISA 检测228例钩体病人血清ompL1基因产物的抗体。结果:与报道的相关序列比较,ltB-ompL1/1和ctB-ompL1/1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%～100%。rLTB-rOmpL1/1和rCTB-rOmpL1/1表达产量均约为细菌总蛋白的10%,主要以包涵体形式存在。rLTB-rOmpL1/1和rCTB-rOmpL1/1均分别能与rOmpL1/1兔抗血清和牛GM₁ 结合。89.7%（87/97）问号钩体野生株含有ompL1基因,87.6%（85/97）问号钩体野生株分别与rOmpL1/1和rOmpL1/2兔抗血清出现效价范围为1:4～1:256的MAT阳性结果。86.8%（198/228）和88.6%（202/228）的病人血清标本分别rOmpL1/1和rOmpL1/2抗体阳性。结论:本文成功地构建了ltB-ompL1/1和ctB-ompL1/1融合基因及其原核表达系统。所表达的rLTB-rOmpL1/1和rCTB-rOmpL1/1融合蛋白有良好的免疫原性和佐剂活性。ompL1是不同问号钩体血清群中广泛存在和高频率表达的基因,且其不同基因型的表达产物有广泛的抗原和抗体交叉。rLTB-rOmpL1/1和rCTB-rOmpL1/1具有成为问号钩体属特异性疫苗抗原的良好前景。更多还原

【Abstract】 Objective:To construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes andto identify immunogenic and adjuvant activities of the expression products,and to detemine frequencies ofompL1 gene carrying and expression in wild strains of L.interrogans and specific antibody in sera fromleptospirosis patients.Methods:PCR using linking primer was applied to construct the fusion genesltB-ompL1/1 and ctB-ompL1/1.By using routine molecular biological techniques,prokaryotic expressionsystems of the two fusion genes were constructed.SDS-PAGE was used to examine expression of thetarget recombinant proteins rLTB-rOmpL1/1 and rCTB-rOrnpL1/1.By using Western Blot andGM₁-ELISA,immunogenic and adjuvant activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1 weremeasured,respectively.PCR and MAT were performed to detect carrying and expression of ompL1 genein 97 wild strains of L interrogans,respectively.Antibodies against products of ompL1 gene in serumsamples of 228 leptospirosis patients were detected by ELISA.Results In comparison with the reportedcorresponding sequences,nucleotide and putative amino acid sequence similarities of ltB-ompL1/1 andctB-ompL1/1 fusion genes were 99.7%-99.9% and 99.5%～100%,respectively.Expression outputs of boththe rLTB-rOmpL1/1 and rCTB-rOmpL1/1,mainly present with inclusion body,were approximate 10% ofthe total bacterial proteins.Both of rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbitanti-rOmpL1/1 serum as well as bovine GM₁,respectively.89.7% of the L.interrogans wild strains（87/97）had ompL1 gene.87.6%（85/97）of the wild strains with the rabbit aniti-rOmpL1/1 or anti-rOmpL1/2 serawere positive for MAT with titers of 1:4～1:256.86.8%（198/228）and 88.6%（202/228）of the patients’serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies,respectively.ConclusionltB-ompL1/1 and ctB-ompL1/1 fusion genes and their prokaryotic expression systems were successfullyconstructed in this study.The two expressed fusion proteins showed qualified immunogenic and adjuvantactivities,ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and the products expressed by different genotypes exhibit high cross-antigenicity andcross-immunoreaction.rLTB-rOmpL1/1 and rCTB-rOmpL1/1 shows fine potential as antigens in L.interrogans genus-specific vaccine.更多还原