问号钩端螺旋体lipL32/1-lipL21-OmpL1/2融合基因原核表达及其产物免疫原性分析  

Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products

作　　者：罗冬娇[1] 邱晓枫[2] 王江[3] 严谨[1] 王海斌[1] 周金成[1] 严杰[3] 

机构地区：[1]杭州师范大学钱江学院,浙江杭州310013 [2]杭州市疾病预防控制中心,浙江杭州310006 [3]浙江大学医学院病原生物学系,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2008年第6期599-604,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省科技厅国际合作重点资助项目(2006C24003)

摘　　要：目的:构建问号钩端螺旋体(简称钩体)lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,优化目的产物表达条件并对表达产物免疫原性进行鉴定。方法:采用连接引物PCR构建lipL32/1-lipL21-OmpL1/2融合基因,并用常规基因工程方法建立其原核表达系统。采用SDS-PAGE及Bio-Rad凝胶图象分析系统,检测目的重组蛋白rLipL32/1-LipL21-OmpL1/2表达量。采用免疫双扩散试验及WesternBlot,鉴定rLipL32/1-LipL21-OmpL1/2的免疫原性。结果:获得了序列正确的lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统E.coliBL21DE-3pET42a-lipL32/1-lipL21-ompL1/2。表达条件优化后的rLipL32/1-LipL21-OmpL1/2产量为37.78mg/L,是优化前的3.7倍。rLipL32/1-LipL21-OmpL1/2兔抗血清免疫双扩效价为1∶4。rLipL32/1-LipL21-OmpL1/2抗血清能识别rLipL32/1-LipL21-OmpL1/2以及rLipL32/1、rLipL21、rOmpL1/2。rLipL32/1-LipL21-OmpL1/2能与问号钩体56601株全菌兔抗血清以及黄疸出血群、流感伤寒群、波摩那群、秋季群问号钩体感染患者血清出现阳性杂交信号。结论:成功地构建了lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,表达产物具有良好的抗原性和交叉免疫反应性,可作为研制通用型问号钩体基因工程疫苗及通用型钩体病血清学检测的抗原。Objective： To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. Methods： PCR using linking primers was applied to construct lipL32/1-lipL21 -OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. Results： lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E. coli BL21DE3^pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1：4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2,rLipL32/1 ,rLipL21 and rOmpL1/ 2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L. interrogans serogroups Icterohaemorrhagiae ,Gri ppotyphosa , Autumnalis and Pomona. Conclusion ： The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

关 键 词：钩端螺旋体 问号 抗原 细菌/分析 聚合酶链反应/方法 属特异性抗原 lipL32基 因/lipL21基因/OmpL1/2 融合基因/构建 原核表达 免疫原性/鉴定 

分 类 号：R377.5[医药卫生—病原生物学]