细粒棘球绦虫TAK1基因ORF的克隆及其蛋白生物信息学分析

Cloning of the ORF of TAK1gene of Echinococcus granulosus and bioinformatic analysis of the protein

目的克隆细粒棘球绦虫(Eg)TAK1基因的开放阅读框(ORF)全长,预测其编码的蛋白EgTAK1的结构特点和生物学功能。方法从新疆株细粒棘球蚴中扩增EgTAK1-ORF全长,克隆至载体pMD20-T上并测序;从NCBI蛋白质数据库中下载EgTAK1的氨基酸序列,利用生物信息学分析软件分别对EgTAK1的理化性质、磷酸化位点、O-糖基化位点、N-糖基化位点、亲/疏水性、保守结构域、跨膜结构域、信号肽序列进行预测;采用MEGA 7.0软件对不同物种来源的TAK1蛋白进行多序列比对并构建系统进化树。采用实时荧光定量PCR检测细粒棘球绦虫在不同发育阶段TAK1基因mRNA的相对表达量。结果EgTAK1基因ORF全长1116 bp,编码371个氨基酸,理论等电点为5.97,不稳定指数为49.59,属于不稳定蛋白;脂肪系数为87.22,总平均亲水系数为-0.291,为亲水性蛋白;EgTAK1蛋白含有64个磷酸化位点,3个O-糖基化潜在位点,4个N-糖基化位点;EgTAK1蛋白属于蛋白激酶C类似(PKC-like)家族;无跨膜结构和信号肽,亚细胞可能定位于细胞膜、细胞质及细胞核内。该蛋白含有7个优势B细胞抗原表位,具有良好的免疫原性。二级结构包含34.77%的α-螺旋,5.12%的β-转角,40.97%的无规卷曲,19.14%的延伸链。TAK1蛋白同源序列比对中与多房棘球绦虫的TAK1同源性最高为83%。实时荧光定量PCR显示,细粒棘球绦虫在不同发育阶段EgTAK1表达有差异,成虫阶段相对表达量最高。结论基因EgTAK1可能是细粒棘球绦虫发育分化的重要调控基因,预测该基因编码的蛋白含有B细胞抗原表位,有可能成为包虫病免疫学预防及药物研制的靶标抗原。

Objective To clone the ORF of gene TAK1 of Echinococcus granulosus,and to predict the structural characteristics and biological functions of EgTAK1 protein.Methods The full length of EgTAK1-ORF was amplified from E.granulosus of Xinjiang strain,cloned into pMD20-T vector and sequenced.The amino acid sequence of EgTAK1 was downloaded from NCBI protein database.The physicochemical properties,phosphorylation sites,O-glycosylation sites,N-glycosylation sites,hydrophilicity/hydrophobicity,conserved domain,transmembrane domain and signal peptide sequence of EgTAK1 were predicted by bioinformatics analysis software.By MEGA 7.0 software,multi-sequence alignment of TAK1 proteins from different species was performed and phylogenetic tree was constructed.The relative expression of TAK1 mRNA in different developmental stages of E.granulosus was detected by real-time quantitative PCR.Results The ORF of EgTAK1 gene was 1 116 bp,encoding 371 amino acids.The theoretical isoelectric point was 5.97 and the instability index was 49.59,which belonged to unstable protein.The fat coefficient was 87.22,and the total average hydrophilic coefficient was-0.291,which was hydrophilic protein;EgTAK1 protein contains 64 phosphorylation sites,3 O-glycosylation potential sites and 4 N-glycosylation sites.EgTAK1 protein belongs to PKC-like family;without transmembrane structure and signal peptide,subcellular localization may be located in cell membrane,cytoplasm and nucleus.The protein contains seven dominant B cell epitopes and has good immunogenicity.The secondary structure contains 34.77% α-helix,5.12% β-turn,40.97% random coil and 19.14% extension chain.In the homology alignment of TAK1 protein,the highest homology with TAK1 of E.multilocularis was 83%.qPCR showed that the expression of EgTAK1 was different at different developmental stages,and the relative expression was the highest at adult stage.Conclusion The gene EgTAK1 may be an important regulatory gene for the development and differentiation of E.granulosus.It is predicted that the protein encoded by this gene contains B cell antigen epitope,which may become the target antigen for immunological prevention and drug development of hydatid disease.