细粒棘球绦虫烯醇酶(EgEnolase)基因的克隆和序列分析

Cloning and Sequence Analysis of Echinococcus granulosus Enolase

目的细粒棘球绦虫烯醇酶基因(EgEnolase)的克隆和所编码的蛋白质的结构、功能以及应用前景分析。方法利用美国国家生物技术信息中心(NCBI,http://www.ncbi.nlm.nih.gov/)的在线分析工具BLASTx和瑞士生物信息学研究所的蛋白分析专家系统(ExPaSy,http://ca.expasy.org/),以及CBS Prediction Servers提供的蛋白序列在线分析工具,结合Vector NTI suite生物信息学分析软件,从GenBank中细粒棘球绦虫的表达序列标签(EST)数据库中发现烯醇酶的5’端和3’端的EST序列,根据预测的编码区两端序列设计引物,从细粒棘球绦虫青海绵羊分离株中用多聚酶链式反应(PCR)方法扩增其基因组序列,PCR产物克隆到T载体,测序并分析序列中的内含子,以内含子两侧序列的合并序列为引物,采用不对称PCR方法去除其中的内含子序列,预测编码蛋白的结构和功能特征,并分析其应用前景。结果细粒棘球绦虫青海绵羊株烯醇酶基因的基因组序列长为1449bp,含有两个长度分别为78bp和69bp的小内含子。该基因编码433个氨基酸;预测其氨基酸序列中含有一段跨膜区(aa104-124),N端在膜外,C端在膜内,恰好分为两个不同的功能域,膜内区是执行酶催化功能的主体,Swiss-Model模建的3D结构显示,膜内区由α螺旋和β折叠相间排列形成桶样结构,底物结合位点、催化中心、Mg2+结合位点等关键位点在空间上紧密靠近,位于桶形结构的中心。该蛋白还有一个潜在的核定位序列aa190-199。该蛋白含有多个T、B细胞表位,且膜外的aa49-57和膜内的aa228-236兼性的T、B细胞表位,线性B细胞表位aa206-213中包含了催化位点Glu210。结论细粒棘球绦虫烯醇酶可能是一个位于虫体皮层表膜具有较好的免疫诊断和疫苗应用前景的膜蛋白,同时还可能进入细胞核调节基因表达。

Objective To clone enolase gene from Echinococcus granulosus and analyze theits structural characteristics of the putative protein and its potential in diagnosis and vaccine development. Methods Comprehensively utilize the online tools provided by the bioinformatics websites such as NCBI （http：//www.ncbi.nlm.nih.gov/）, ExPaSy （http：//www.ncbi.nlm.nih.gov/）, and CBS Prediction Servers （http：//www.cbs.dtu.dk/serviees/） and incorporate Vector NTI suite 8.0 and DNAstar software package, the 5＇ and 3＇ expression tags （EST） of E.granulosus enolase were founddiscovered from GenBank database by BLASTx. According to the predicted coding sequence, primers were designed and were used to amplify the genomic sequence of E.granulosus strain isolated from sheep in Qinghai province of China by polymerase chain reaction （PCR）. The product of PCR was cloned into TA plasmid and sequenced to analyze the intron sequence. The intron sequences were excised by unsymmetrical PCR with combined flank sequences of introns as primers. The structural, functional and immunological characteristics of putative amino acid sequence were predicted by bioinformatics analysis. Results The genomie sequence of enolase from E.granulosus sheep strain isolated from Qinhai was 1 449 bp in length and contains two smalllittle introns of 78 bp and 69 bp respectively. The complete coding sequence encodes 433 amino acids and contains a transmembrane region aa104-124, with the N terminus facing outside and C terminus inside. The inside part is probablyquite the functional domain and Swiss-Model modulatedpredicted its 3D structure is comprised of alternatively arranged α helix-β sheet and forms a barrel, with the key sites including substrate binding region, the active sites, Mg2. binding sites closely located in the centre of barrel. The protein contains a possible nuclear location sequence aa190-199 and several linear B cell epitopes and CTL T cell epitopes, and the outside epitope aa49-57 and inside epitope aa228-236 are faeuhative T cell and B cell epitopes and the linear B cell epitope aa206-213 contains the active centre site Glu230. Conclusion E.granulosus enolase is probablye a tegumental membrane protein, which can enter nucleus to regulate gene expression, with potential asfor a diagnostic and vaccine antigen and meanwhile can enter nucleus to regulate gene expression.