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目的用生物信息学方法识别欧猥迭宫绦虫(Spirometra erinaceieuropaei,S.e)乳酸脱氢酶(LDH)全长cD-NA序列,预测其编码蛋白的结构与功能。方法用NCBI、EMBI、Expasy等在线网站对序列结构域、功能域等进行分析以识别基因,预测其蛋白序列理化参数、信号肽、抗原表位、拓扑结构等,三级结构同源建模应用Vector软件进行同源序列比对、构建进化树及三级结构模型分析。结果目标序列具有完整开放阅读框、L-LDH结构域及功能域、polyA,确定为LDH全长cDNA,命名为SeLDH(GU 121 968);该序列编码338氨基酸残基(aa),预测等电点为6.38,分子量为36 028.6Da;与华支睾吸虫、日本血吸虫及人LDH的相似性分别为60%、59%及55%;有5个可能跨膜区域;主要线性表位中的185～191 aa、223～235 aa及328～338 aa与人LDH相同区域差异明显;表位104～111 aa与人LDH相同区域有1 aa的差异,其上有NAD及底物结合位点,关键催化位点112 R紧邻该区域;三级结构模型显示其在蛋白表面形成环状结构,3个关键催化位点及NAD、丙酮酸结合位点在该环状结构上或周围形成催化中心。结论获得SeLDH全长cDNA序列;该蛋白可能是膜蛋白;是理想的免疫诊断、药物作用及疫苗靶分子。
Objective To identify the full-length cD-NA sequence of lactate dehydrogenase (LDH) from Spirometra erinaceieuropaei (S.e.) using bioinformatics methods and predict its structure and function. Methods The sequence domains and functional domains were analyzed by NCBI, EMBI, Expasy and other online websites to identify the genes, and to predict the physical and chemical parameters of protein sequences, signal peptides, epitopes and topologies. The application of tertiary structure homology modeling Vector software for homologous sequence alignment, construction of evolutionary tree and tertiary structure model analysis. Results The target sequence contained a complete open reading frame, L-LDH domain and its functional domain, polyA, which was identified as LDH full-length cDNA and named SeLDH (GU 121 968). The sequence encoded 338 amino acid residues (aa) Point 6.38, molecular weight 36 028.6Da; similarities to Clonorchis sinensis, Schistosoma japonicum and human LDH were 60%, 59% and 55% respectively; there were 5 possible transmembrane regions; 185 of the major linear epitopes ~ 191 aa, 223 ~ 235 aa and 328 ~ 338 aa were significantly different from those in human LDH. The difference between epitopes 104 ~ 111 aa and human LDH in the same region was 1 aa with NAD and substrate binding sites The catalytic site 112R is immediately adjacent to the region; the tertiary structure model shows that it forms a ring structure on the protein surface, with three key catalytic sites and NAD, pyruvate binding sites forming catalytic centers on or around the ring structure. Conclusion The full-length cDNA sequence of SeLDH is obtained. This protein may be a membrane protein and is an ideal immunodiagnosis, drug action and vaccine target molecule.