论文部分内容阅读
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 the EgEnolase gene of Echinococcus granulosus and analyze its structure, function and application prospect. METHODS: BLASTx, an online analysis tool at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), and a protein analysis expert system at the Swiss Institute of Bioinformatics (ExPaSy, http: // ca.expasy.org/) and on-line analysis of protein sequences provided by CBS Prediction Servers, enolase was identified from the expressed sequence tag (EST) database of Echinococcus granulosus in GenBank using Vector NTI suite bioinformatics analysis software The 5 ’end and 3’ end of the enzyme were sequenced and the primers were designed according to the two ends of the predicted coding region. The genomic sequence was amplified by polymerase chain reaction (PCR) from the Echinococcus granulosus isolates. PCR The products were cloned into the T vector, sequenced and analyzed for introns. The intron sequences were sequenced by using the combined sequences on both sides of introns as primers, and the intron sequences were predicted by asymmetric PCR to predict the structure and function of the encoded proteins. And analyze its application prospects. Results The genomic sequence of enolase gene of Echinococcus granulosus was 1449 bp in length and contained two small introns with length of 78 bp and 69 bp, respectively. The gene encodes 433 amino acids; it is predicted that the amino acid sequence contains a transmembrane region (aa104-124). The N-terminal is outside the membrane and the C-terminal is within the membrane, which is divided into two different functional domains. The main structure of enzyme catalysis, the 3D structure modeled by Swiss-Model, showed that the intra-membrane region formed a barrel-like structure by the arrangement of α-helix and β-sheet, and the key sites such as substrate binding site, catalytic center and Mg2 + binding site were Close in space, in the center of the barrel structure. The protein also has a potential nuclear localization sequence aa190-199. The protein contains multiple T, B cell epitopes, and extracellular aa49-57 and membrane aa228-236 facultative T, B cell epitopes, linear B cell epitope aa206-213 contains a catalytic site Glu210. Conclusion Enolase of Echinococcus granulosus may be a membrane protein with better immunodiagnosis and vaccine application prospect in the epidermis of the parasites and may also enter the nucleus to regulate the gene expression.