细粒棘球绦虫原头蚴蛋白表达谱分析

摘要

旨在探索细粒棘球绦虫原头蚴蛋白表达特性,揭示在此发育阶段虫体主要的功能蛋白以及信号通路,为揭示虫体的发育调控、筛选疫苗以及药物靶标奠定基础.本试验采用LC-MS/MS技术对虫体原头蚴表达的蛋白质进行研究,然后对获得数据进行GO、KOG以及KEGG等分析;最后采用qRT-PCR和原位杂交技术对鉴定得到的Wnt信号通路的关键基因β-catenin进行了差异表达和组织定位研究.结果显示,本研究共鉴定得到7 172个肽段,1 197个蛋白质,其中包括多个潜在的抗原蛋白质,如四跨膜蛋白超家族、6-磷酸葡萄糖酸脱氢酶、表皮抗原蛋白和烯醇酶等.信号通路分析结果显示其中632个蛋白参与276个调控通路,诸如与虫体发育紧密相关的Wnt、Notch、Hedgehog、NF-κB、cAMP以及胆酸盐信号通路.对β-catenin的验证结果显示,此基因分布于细粒棘球绦虫原头蚴顶突的小刺以及散在的细胞中,同时其相对转录量在体外培养0～10 d的过程中呈递减趋势.综上,本研究解析了细粒棘球绦虫原头蚴的蛋白质表达元件,揭示了原头蚴主要的调控信号通路,为虫体的生长发育机制以及包虫病的有效防控提供了理论依据.%The aim of this study was to explore the protein expression characteristics of protoscoleces,to reveal the main functional proteins and signaling pathways during the development of protoscoleces in Echinococcus granulusos,to lay the foundation for the mechanisms of development regulation of protoscoleces and screening target genes for vaccines and drugs.The LC-MS/ MS technology was used to analyze the proteins expressed in the protoscoleces,then the proteins were analyzed based on GO,KOG and KEGG.Finally,the qRT-PCR and in situ hybridization were used to analyze the different expression and localization of the β-catenin which is the key gene of the Wnt signaling pathway.The results showed that the present study identified 7 172 peptides,1 197 proteins,including multiple potential antigenic proteins,such as tetraspanin superfamily,6-phosphogluconate dehydrogenase,enolase and epidermal antigenic proteins.Signal pathway analysis showed that 632 of these proteins involved in 276 pathways,such as Wnt,Notch,Hedgehog,NF-κB,cAMP and bile acid signaling pathways closely related to development of E.granulosus.The in situ hybridization analysis showed that the β-catenin was mainly distributed in the hooks and dispersed cells of the protoscoleces.Meanwhile,the relative transcription of β-catenin was gradually decreased during 0 to 10 days in vitro culture.In summary,this study analyzed the protein expression elements of protoscoleces,revealed the main regulatory pathways of protoscolex development,and provided a theoretical basis for the growth mechanisms of protoscoleces and effective prevention of hydatid disease.