细粒棘球绦虫CD151蛋白的生物信息学分析及其编码基因在不同发育阶段的表达

Gene sequencing and analysis of the expression of CD151 in different developmental stages of Echinococcus granulosus

目的分析细粒棘球绦虫四跨膜蛋白家族成员CD151(Eg-CD151)分子特性和各发育阶段表达水平,为研发包虫病疫苗奠定基础。方法从NCBI数据库下载Eg-CD151氨基酸序列,利用ProtParam、NetPhos 3.1 Server、GPS-SUMO 2.0、Euk-mPLoc 2.0、SOPMA、SWISS MODEL、SignalP 4.1 Server、TMHMM、SMART、Antibody Epitope Prediction在线生物信息学软件预测Eg-CD151蛋白的理化性质、磷酸化位点、棕榈酰化修饰位点、亚细胞定位、二级结构、三维结构、跨膜区域、结构域、B细胞抗原表位;利用DNAMAN和MEGA进行同源序列分析并建立系统发育进化树。利用实时荧光定量PCR检测不同发育阶段Eg-CD151 mRNA相对表达量。结果Eg-CD151蛋白由170 aa组成,为一种不稳定的跨膜蛋白,无信号肽,含2个跨膜区,分别位于6-28、136-158 aa,3个B细胞抗原表位位于非跨膜区域;存在14个磷酸化位点,包含3个酪氨酸磷酸化位点、5个苏氨酸磷酸化位点、6个丝氨酸磷酸化位点;含一个棕榈酰化位点(139-143 aa);二级结构主要为α-螺旋,占60.00%。同源序列比对显示,Eg-CD151与其终末宿主犬、赤狐及中间宿主牛的CD151进化关系较远。实时荧光定量PCR检测原头蚴、成虫两个发育阶段Eg-CD151 mRNA相对表达量差异无统计学意义(P>0.05)。结论生物信息学分析Eg-CD151存在多个B细胞抗原表位,可作为包虫病诊断及疫苗候选抗原。

Objective To analyze the molecular characteristics and differential expression of Eg-CD151, a member of the tetraspanin family, in different developmental stages of Echinococcus granulosus in order to provide a foundation for the development of echinococcosis vaccines. Methods The amino acid sequence of Eg-CD151 was downloaded from the NCBI database. ProtParam, NetPhos 3.1 Server, GPS-SUMO 2.0, Euk-mPLoc 2.0, SOPMA, SWISS MODEL, SignalP 4.1 Server, TMHMM, SMART, and Antibody Epitope Prediction were used to predict the physicochemical properties, phosphorylation sites, palmitoyl modification sites, subcellular localization, secondary structure, tertiary structure, transmembrane regions, domains, and B-cell epitopes of the Eg-CD151 protein. DNAMAN and MEGA were used to perform homologous sequence analysis and to construct a phylogenetic tree. The relative levels of Eg-CD151 mRNA expression during different developmental stages were detected using quantitative PCR. Results The Eg-CD151 protein, consisting of 170 amino acids, is an unstable transmembrane protein with no signal peptides. It contains two transmembrane regions, located at amino acids 6-28 and 136-158. Three B-cell epitopes were located in the non-transmembrane region. The protein had 14 phosphorylation sites, including 3 tyrosine phosphorylation sites, 5 threonine phosphorylation sites, and 6 serine phosphorylation sites. It contains a palmitylation site(amino acids 139-143). The secondary structure of Eg-CD151 mainly consisted of α-helices(60.00%). Homologous sequence alignment indicated that Eg-CD151 was distantly related to CD151 in its definitive hosts, dogs and foxes, and its intermediate host, cattle. Quantitative PCR indicated that there were no significant differences in the relative expression of Eg-CD151 mRNA between the two developmental stages of protoscoleces and adults(P>0.05). Conclusion Bioinformatic analysis indicated that Eg-CD151 had multiple B-cell epitopes. Eg-CD151 could potentially be used as an antigen for diagnosis of and vaccines against hydatid disease.