问号钩端螺旋体T和B细胞表位联合基因的原核表达及其产物免疫性分析

目的 构建问号钩端螺旋体(简称钩体)LipL32、OmpL1和LipL21蛋白的优势T-和B-细胞联合表位融合基因及其原核表达系统,并对表达产物的免疫原性进行鉴定.方法 人工合成多表位联合基因并构建其原核表达系统.采用SDS-PAGE检测重组蛋白；采用MAT检测重组蛋白兔抗血清与我国钩体标准参考株的凝集效价；Western blot和ELISA检测重组蛋白的免疫原性.结果 获得了多表位融合基因并构建了原核表达系统.表达产物的相对分子质量约为23×103,且主要以可溶性形式存在；重组蛋白兔抗血清免疫双扩散效价为1∶8,该抗血清能与我国15群的钩体标准参考株发生凝集反应,ELISA证明该重组蛋白能检测不同群型钩体感染患者血清中的抗钩体抗体.结论 成功构建了包含钩体LipL32、OmpL1和LipL21蛋白的优势T和B细胞联合表位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血清学检测的抗原.

Expression of T and B cells combined epitopes protein of Leptospira interrogans and the immunity analysis

Objective To construct the T-cells and B-cells combined epitope peptide gene based on the LipL32,OmpL1 and LipL21 protein from Leptospira interrogans and E.coli expression system,and better understanding of the immunological activity of the recombinant protein. Methods The immunodomaint T- and B-cells combined epitopes of LipL32,OmpL1 and LipL21 were identified and used to synthetic a new gene and then construct its prokaryotic expression system.The expression of recombinant protein was determined by SDS-PAGE; MAT was used to determine the titer of the antiserum to L. interrogans standard strains of China ; Western blot and ELISA were used to identify the immunity activity of the recombinant protein.Results The synthetic gene was effectively expressed in E.coli BL21 ( DE3 ) strain and mainly presented in dissoluble protein.Western blot result showed that the expression protein react well with the antibodies from immunized rabbit by Leptospira or recombinant protein.ELISA and MAT results showed that the multiepitope protein could cross-react with different serogroup or serovar of Leptospira.Conclusion In this study,we successfully constructed the recombinant T- and B-cells combine epitope gene of leptospires and expressed it in E.coli.The recombinant protein had a good immune activity,and could cross-reacted with antibodies from different serogroups Leptospira infected patients.