欧猥迭宫绦虫乳酸脱氢酶新基因识别及其结构与功能预测

Identification of a novel gene encoding lactate dehydrogenase from Spirometra erinaceieuropaei and prediction for its structure and function

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 cDNA sequence encoding lactate dehydrogenase（LDH） from adult Spirometra erinaceieuropaei（S.e.） and to predict the structure and function of the protein.Methods Some online websites such as NCBI,EMBI,Expasy and a software packages Vector NTI were exploited to analyze the sequence bioinformatics,including the open reading frame（ORF）,conserved structure and functional domain,multi-sequence homological alignment,phylogenetic analysis,topological prediction,homology modeling of tertiary structure,and antigenic epitope analysis.Results The target sequence had a full ORF,typical L-LDH structure domain,full conserved domain and polyA.It was confirmed as a full length cDNA encoding LDH of S.e.and then named as SeLDH（GU121968）.The sequence encodes a protein of 338 amino acids with the predicted molecular weight of 36 028.6Da and isoelectric point of 6.38.Compared with the LDHs of Clonorchis sinensis,Schistosoma japonicum and human,the similarity of SeLDH is 60%,59%,and 55%,respectively.In the protein,5 transmembrane regions were found and 185-191aa,223-235aa,328-338aa of the 5 major epitopes were presented with significant difference compared to the same region of human LDH.There is only 1aa difference between epitope 104-111aa of SeLDH and that of human,which is an important function region containing 2 NAD binding sites and 1 substrate binding site and the key catalytic residue 112R is close to the region.Tertiary structure demonstrates that 104-111aa is on the surface of the protein and forms a substrate binding loop,three key catalytic sites and NAD,pyruvate binding sites formed a catalytic center near the loop.Conclusion The full length cDNA sequence of a novel gene SeLDH was obtained.The bioinformatics implies that SeLDH might be a transmembrane protein and an ideal molecule for immunodiagnosis,vaccine and drug reaction.