曼氏迭宫绦虫中亮氨酸氨基肽酶基因的生物信息学分析和转录水平检测

Bioinformatics analysis,transcriptional level detection of leucine aminopeptidases from Spirometra mansoni

目的检测曼氏迭宫绦虫的3个亮氨酸氨基肽酶亚类(leucine aminopeptidases of Spirometra mansoni,Sm LAPs)基因在虫体不同发育阶段的转录水平,为进一步病原诊断研究奠定基础。方法运用生物信息学相关软件对3个亮氨酸氨基肽酶亚类编码蛋白的氨基酸序列的功能区域、免疫学特征、信号肽和跨膜区预测分析。应用Real time PCR方法检测Sm LAPs在曼氏迭宫绦虫的成节、孕节和裂头蚴阶段的转录水平。结果对Sm LAPs编码氨基酸序列的保守区域进行比对,Sm LAPa与Sm LAPb、Sm LAPc的氨基酸序列一致性均为38%,Sm LAPb和Sm LAPc的一致性也只有44%。多功能位点分析发现,Sm LAPs均含有底物结合位点、锌离子结合位点和多肽结合位点。在B细胞线性表位预测结果显示,Sm LAPa有13个、Sm LAPb有14个、Sm LAPc有13个。T细胞表位预测中,Sm LAPa有12个、Sm LAPb有13个、Sm LAPc有14个。3个Sm LAPs亚类序列中均含有一个强烈的信号肽,并且均没有跨膜区域。在虫体各个发育阶段的转录水平检测中,在成节阶段,Sm LAPb的转录水平是Sm LAPa的1.34倍,Sm LAPc的转录水平是Sm LAPa的46.53倍。在孕节阶段,Sm LAPb的转录水平是Sm LAPa的1.96倍,Sm LAPc的转录水平是Sm LAPa的56.89倍。在裂头蚴阶段,只有Sm LAPc有转录,没有检测到Sm LAPa和Sm LAPb的转录。结论在3个Sm LAPs亚类基因中,Sm LAPc是裂头蚴病更有价值的免疫诊断分子。

Objective To detect transcription levels of 3 leucine aminopeptidases from Spirometra mansoni in different developmental stages of the parasite,which lays a foundation for pathogenic diagnosis of further study.Methods Using bioinformatics software,the functional region,immunological characteristics,signal peptide and transmembrane domain of Sm LAPs were analyzed.The transcriptional levels of Sm LAPs at mature proglottid,gravid proglottid and sparganum stages were detected.Results In the alignment of conserved domains of Sm LAPs,the amino acid sequence identities of Sm LAPa were 38%compared with Sm LAPb and Sm LAPc.The identity between Sm LAPb and Sm LAPc was 44%.Multi-functional sites analysis found that Sm LAPs contained substrate binding sites,zinc ion binding sites and peptide binding sites.In the prediction of B cell epitopes,Sm LAPa and Sm LAPc had 13 B cell epitopes,while Sm LAPb had 14 B cell epitopes.The T cell epitope prediction showed that Sm LAPa,Sm LAPb and Sm LAPc contained 12,13 and 14 T cell epitopes,respectively.3 Sm LAPs had strong signal peptides.In addition,there were no transmembrane regions in the amino acid sequences.The transcriptional level of Sm LAPb was 1.34 times and that of Sm LAPc was about 46.53 times compared with that of Sm LAPb at the mature proglottid.In the gravid proglottid stages,the results of Sm LAPb and Sm LAPc were 1.96 times and 46.53 times respectively,while Sm LAPa was at the background value.Interestingly,at the sparganum stage,Sm LAPc transcription expression was detected,whereas the transcriptional levels of Sm LAPa and Sm LAPb were low.Conclusions Sm LAPc might be a valuable diagnostic molecule for Spirometra mansoni infection.