钩端螺旋体OmpL1抗原表位预测、克隆和表达  

Cloning and expression of the gene fragment encoding for epitope of outer membrane protein OmpL1 from Leptospira

作　　者：杨榆玲[1] 褚嘉祐[1] 郭晓奎[2] 石铁流[3] 黄小琴[1] 俞建昆[1] 

机构地区：[1]中国医学科学院,中国协和医科大学,医学生物学研究所,昆明650118 [2]上海第二医科大学病原生物学教研室,上海200025 [3]中科院上海生命科学信息中心,上海200032

出　　处：《中国人兽共患病杂志》2005年第1期27-31,共5页Chinese Journal of Zoonoses

基　　金：国家高技术研究发展计划 [2 0 0 3AA2 2 3 0 3 1] ;国家自然科学基金(3 0 3 70 0 71和 3 0 3 0 0 197)

摘　　要：目的　应用生物信息学方法预测钩体外膜蛋白OmpL1的表位 ,结合基因工程手段进行表位重组、表达和分离纯化。方法　用预测程序ProPred和ANTIGENIC预测OmpL1的表位 ,PCR合成重组表位基因片段 ,克隆PCR产物构建表达质粒 pGEX/Omp Omp ,测序验证。对含有该质粒的大肠杆菌BL2 1(DE3)进行诱导表达 ,表达产物westernblot分析并纯化融合蛋白。结果　预测到 2个既具有MHC结合肽特性又具有B细胞表位特征的肽段。重组表位基因序列与理论设计完全一致。IPTG诱导BL2 1(DE3)中高效表达Mr约 30 0 0 0的融合蛋白 ,纯化后蛋白纯度 >90 %。结论　成功构建原核表达质粒pGEX/Omp Omp ,并进行含重组表位的GST融合蛋白的分离纯化 ,为OmpL1的表位研究和应用于亚单位疫苗奠定了基础。The epitope of OmpL1 from Leptospira was predicted by bioinformation methods, such as prediction programs of ProPred and ANTIGENIC, and the recombinant epitope gene was constructed by PCR.The PCR product was cloned to construct the expression vector pGEX/Omp Omp.After the gene was sequenced, the E.coli host strain BL21 (DE3) containing recombinant plasmid was induced to express the fusion protein, and this protein was purified.It was found that two peptide fragments with both the MHC binding peptide property as well as the B cell peptide characters were predicted in this way. The sequences of the recombinant epitopes were just the same as those designed, and the host strain BL21 (DE3) could express a Mr. 30,000 fusion protein after induction with IPTG.Its purity was over 90%.It concludes that the expression vector pGEX/Omp Omp is successfully constructed in the present study. This provide for a base for the further studies on the epitopes of OmpL1 and for the use as the recombinant epitope in subunit vaccine.

关 键 词：OMPL1 抗原表位 钩端螺旋体 

分 类 号：R377[医药卫生—病原生物学]