细粒棘球绦虫腺苷酸活化蛋白激酶AMPKβ基因克隆及生物信息学分析

Cloning and bioinformatic analysis of the AMP-activated protein kinaseβgene fromEchinococcus granulosus sensu stricto

目的克隆细粒棘球绦虫腺苷酸活化蛋白激酶β(AMP-activated protein kinaseβof Echinococcus granulosus sensu stricto,EgAMPKβ)基因全长序列,并进行生物信息学分析。方法提取细粒棘球绦虫原头蚴总RNA,利用PCR技术克隆EgAMPKβ基因全长序列,并采用生物信息学技术对该基因编码蛋白的理化性质、磷酸化位点、抗原表位、结构域、二级结构、三级结构、多序列分析和亲缘性比较等进行生物信息学分析。结果EgAMPKβ的cDNA全长981 bp,编码326个氨基酸,相对分子质量为35.23585×10^(3),属于亲水性蛋白,无信号肽序列和跨膜区,具有22个丝氨酸磷酸化位点,15个苏氨酸磷酸化位点,含有5个酪氨酸磷酸化位点;4个B细胞抗原表位,具有碳水化合物结合模块(Carbohydrate-binding module,CBM)和αγ亚单位相互作用域(αγ-subunits interaction domain,αγ-SID)。EgAMPKβ基因与多房棘球绦虫和人AMPKβ基因序列相似性分别为98.47%和39.57%,与多房棘球绦虫、微口膜壳绦虫同属于绦虫纲,与人、猩猩和小鼠等哺乳纲动物的进化关系较远。结论成功克隆了EgAMPKβ基因,生物信息学分析其编码蛋白具有保守的功能结构域,对细粒棘球绦虫的能量代谢具有调控作用,为进一步了解细粒棘球绦虫能量代谢机制及其新型抗包虫病药物靶点开发提供了研究基础。

Objective To clone the full-length sequence of the AMP-activated protein kinaseβgene of Echinococcus granulosus sensu stricto(E.granulosus ss)(EgAMPKβ),and to bioinformatically analyze the gene.Methods Total RNA of E.granulosus ss protoscoleces was extracted,and the full-length sequence of the EgAMPKβgene was cloned using PCR.Bioinformatic analysis,including multiple sequence analysis and comparison of its affinity,was performed to determine aspects of the gene such as its physicochemical properties,phosphorylation sites,subcellular localization,epitopes,structural domains,secondary structure,and tertiary structure.Results The cDNA of EgAMPKβis 981 bp in length,it encodes 326 amino acids,and it has a molecular weight of 35.23585×10^(3).It is a hydrophilic protein with no signal peptide sequences or transmembrane regions.It has 22 serine phosphorylation sites,15 threonine phosphorylation sites,5 tyrosine phosphorylation sites,and 4 B-cell epitopes.EgAMPKβhas a carbohydrate-binding module(CBM)and anαandγsubunit interaction domain(αγ-SID).EgAMPKβis 98.47%similar to E.multilocularis and 39.57%similar to Homo sapiens AMPKβ.EgAMPKβis evolutionarily related to E.multilocularis and Hymenolepis microstoma in the class Taenia,but it is distantly related to Homo sapiens,Pan paniscus,Mus musculus,and other mammals.Conclusion The EgAMPKβgene was successfully cloned,and this gene was found to contain a conserved functional domain that regulates the energy metabolism of E.granulosus ss.These findings provide a basis for further understanding of the mechanism of energy metabolism in E.granulosus sensu lato and the development of new anti-echinococcosis drug targets.