| 摘要: |
| 【目的】分析β-连环蛋白（β-catenin）在不同发育阶段日本血吸虫体内的表达特征，为研究Wnt/β-catenin信号通路对虫体发育的调控奠定基础。【方法】以7 d童虫cDNA为模板，通过RACE技术获得日本血吸虫β-catenin基因全长cDNA序列。采用实时定量PCR分析该基因mRNA在不同发育阶段日本血吸虫体内表达水平的变化。以含有多个抗原表位的β-catenin部分cDNA序列为模板，构建原核pET28a-β-catenin-310重组质粒，表达β-catenin蛋白。以纯化的原核表达产物为抗原，免疫BALB/c小鼠制备抗血清，以此抗血清为一抗，通过Western blotting分析β-catenin蛋白在不同发育阶段日本血吸虫体内表达量的变化。【结果】β-catenincDNA（GenBank登陆号GU570442）序列全长2 354 bp，含完整的开放阅读框，编码671个氨基酸。β-catenin基因mRNA表达水平在虫卵中最高，23 d雌虫次之，在13 d童虫和23 d雄虫中也较高，约是23 d雌虫的一半，在其他发育阶段mRNA的表达水平相对较低。构建的重组质粒以包涵体形式表达β-catenin重组蛋白，该蛋白有很好的免疫原性。在日本血吸虫不同发育阶段β-catenin蛋白表达量的变化趋势与β-catenin mRNA表达水平的变化基本一致。【结论】不同发育阶段日本血吸虫β-catenin基因mRNA表达水平与β-catenin蛋白表达水平变化趋势基本一致；结合日本血吸虫发育特征，推测Wnt/β-catenin信号通路可能对虫体的器官分化、虫卵的形成及卵胚发育等过程起着重要的调控作用。 |
| 关键词: 日本血吸虫 β-catenin基因 克隆表达 Wnt信号通路 |
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| 基金项目:国家自然科学基金项目(30600450)；国家重点基础研究发展规划(“973”计划)项目(2007CB513108)；中央级公益性科研院所基本科研业务费项目(2008JB02，2010JB07) |
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| Cloning of β-catenin gene from Schistosoma japonicum and its stage specific mRNA and protein level expression |
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| Abstract: |
| 【Objective】The research was to analyze the mRNA and protein level of β-catenin from Schistosoma japonicum at specific developmental stages and further study the role of Wnt/β-catenin signaling pathway in the development of schistosomes.【Method】RACE was applied to acquire full length cDNA of β-catenin from 7 d schistosomula cDNA.The mRNA expression profiles of β-catenin were determined by real time quantitative PCR (qRT-PCR) using the template cDNAs isolated from parasites of specific stages.The partial cDNA with several epitopes of β-catenin was then subcloned into pET28a(+) vector and transformed into competent Escherichia coli BL21(DE3) for expression.Recombinant protein was purified,renaturated,and used as antigen for anti-serum production.With S.japonicum α-tubulin as the endogenous control,the protein expression of β-catenin was analyzed by Western blotting using protein extracted from parasites of specific stages.【Result】The β-catenin cDNA containing complete Open Reading Frame (ORF) was obtained with 2 354 bp,encoding 671 amino acids with molercular weight 75.69 ku.High levels of β-cateninmRNA were detected in eggs and 23-day female worms,the mRNA expression at 13-day schistosomulae and 23-day male worms were both nearly half of the 23-day female level,while worms at other stages had relative low β-catenin mRNA expression.The β-catenin protein was expressed by the recombinant plasmid pET28a-β-catenin-310 in the form of cytorrhyctes,and the protein had relative high immunogenicity.The protein expression of Sj β-catenin from parasites of specific stages was similar to mRNA expression profiles of S.japonicum β-catenin.【Conclusion】The study elucidated the expression of β-catenin from S.japonicum at specific developmental stages,combined with the development of S.japonicum,so it could be concluded that the canonical Wnt/β-catenin signaling pathway might be involved in regulating specific organ differentiation,egg formation and development. |
| Key words: Schistosoma japonicum β-catenin gene cloning and expression Wnt signaling pathway |
