问号钩端螺旋体lipL32/1-lipL41/1融合基因原核表达系统的构建及其应用  

Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1fusion gene

作　　者：罗冬娇[1] 严杰[1] 毛亚飞[1] 李淑萍[1] 罗依惠[1] 李立伟[1] 

机构地区：[1]浙江大学医学院病原生物学教研室

出　　处：《浙江大学学报（医学版）》2005年第1期27-32,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金项目 (39970 6 78)

摘　　要：目的 :构建问号钩端螺旋体 (简称钩体 ) lip L 32 / 1- lip L 4 1/ 1融合基因及其原核表达系统 ,了解钩体野生株 lip L32和 lip L4 1基因携带和表达情况及钩体患者的血清抗体水平。方法 :采用连接引物 PCR构建 lip L32 / 1- li-p L4 1/ 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 r L ip L32 / 1- r L ip L4 1/ 1表达情况。采用 Western blot鉴定 r Lip L32 / 1- r Lip L4 1/ 1的免疫原性。采用 PCR和 MAT分别检测 97株问号钩体野生株 lip L32和 lip L4 1基因及其表达情况。采用 EL ISA检测 2 2 8例钩体患者血清 lip L32和 lip L4 1基因产物的抗体。结果 :与报道的相关序列比较 ,lip L 32 / 1- lip L 4 1/ 1融合基因核苷酸和氨基酸序列相似性分别为 99.9%和99.8%。目的重组蛋白 r Lip L32 / 1- r Lip L4 1/ 1表达产量约为细菌总蛋白的 10 % ,主要以包涵体形式存在。r L ip L32 /1和 r Lip L4 1/ 1兔抗血清均能与 r L ip L32 / 1- r L ip L4 1/ 1结合。 97.9%和 87.6 %问号钩体野生株分别有 lip L32和 li-p L4 1基因。95 .9%和 84 .5 %问号钩体野生株分别与 r Lip L32 s和 r Lip L4 1s兔抗血清出现效价 ,为 1∶ 4～ 1∶ 12 8的MAT阳性结果。分别有 94 .7%～ 97.4 %和 78.Objective: To construct lipL32/1 lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients. Methods: lipL32/1 lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques.SDS PAGE was used to examine expression of the target recombinant protein rLipL32/1 rLipL41/1.Immunogenicity of rLipL32/1 rLipL41/1 was identified by Western blot.PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains.Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method. Results : The homogeneity of nucleotide and putative amino acid sequence of lipL32/1 lipL41/1 fusion gene were 99 9% and 99 8% in comparison with the reported sequences.Expression output of the target recombinant protein rLipL32/1 rLipL41/1,mainly present in inclusion body,accounted for 10% of the total bacterial proteins.Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1 rLipL41/1.97 9% and 87.6% of the L.interrogans wild strains had lipL32 and lipL41 genes,respectively.95 9% and 84 5% of the wild strains were positive for MAT with titers of 1∶4~1∶128 using rabbit anti rLipL32s or anti rLipL41s sera,respectively.94 7%~97 4% of the patients' serum samples were positive for rLipL32s antibodies,while 78 5%~84 6% of them were rLipL41s antibodies detectable. Conclusion: lipL32/1 lipL41/1 fusion gene and its prokaryotic expression system were successfully constructed.The expressed fusion protein had qualified immunogenicity.Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.int

关 键 词：钩端螺旋体 问号 lipL32基因 lipL41基因 序列同源性 核酸 序列同源性 氨基酸 基因表达 克隆 分子 lipL32/1-lipL41/1融合基因/免疫学 

分 类 号：R346[医药卫生—基础医学]