Objective:To identify full length cDNA sequence of lactate dehydrogenase(LDH) from adult Echinococcus granulosus(E.granulosus) and to predict the structure and function of its encoding protein using bioinformatics methods.Methods:With the help of NCBI,EMBI, Expasy and other online sites,the open reading frame(ORF),conserved domain,physical and chemical parameters,signal peptide,epitope,topological structures of the protein sequences were predicted and a homology tertiary structure model was created:Vector NT1 software was used for sequence alignment,phylogenetic tree construction and tertiary structure prediction. Results:The target sequence was 1 233 bp length with a 996 bp biggest ORF encoding 331 amino acids protein with typical L-LDH conserved domain.It was confirmed as full length cDNA of LDH from E.granulosus and named as EgLDH(GenBank accession number:HM748917).The predicted molecular weight and isoelectric point of the deduced protein were 3 5516.2Da and 6.32 respectively.Compared with LDHs from Taenia solium,Taenia saginata asiatica,Spirometra erinaceieuropaei.Schistosoma japonicum,Clonorchis sinensis and human,it showed similarity of 86% ,85% ,55% ,58% ,58% and 53% ,respectively.EgLDH contained 3 putative transmembrane regions and 4 major epitopes(54aa-59aa.81aa-87aa,97aa-102aa,307aa-313aa),the latter were significant different from the corresponding regions of human LDH.In addition,some NAD and substrate binding sites located on epitopes 54aa-59aa and 97aa-102aa,respectively.Tertiary structure prediction showed that 3 key catalytic residues 105R,165D and 192H forming a catalytic center near the epitope 97aa-102aa,most NAD and substrate binding sites located around the center.Conclusions:The full length cDNA sequences of EgLDH were identified.It encoded a putative transmembrane protein which might be an ideal target molecule for vaccine and drugs.
Gang LuYajun LuLihua LiLixian WuZhigang FanDazhong ShiHu WangXiumin Han
目的:构建细粒棘球绦虫(E.g.)成虫全长cDNA质粒文库,并检测文库质量。方法:提取E.g.成虫mRNA,应用SMART方法构建pBluescript II SK全长cDNA质粒文库,检测文库的重组率及容量;用载体克隆位点两端的引物进行PCR扩增,检测插入片段大小。随机挑选阳性重组克隆5′端测序,归并unigene,计算unigene得率,全长性判定主要根据同源全长基因5'末端进行比较判定并计算全长率。结果:成功构建E.g.成虫全长cDNA质粒文库。文库的重组率为95.04%,库容量1.13×106;平均插入片段长度约为1.2 kb。24个随机阳性克隆测序,unigene比例为69.57%,全长性比率为64.71%。结论:成功构建E.g.成虫全长cDNA质粒文库,文库质量良好。
