日本血吸虫Wnt受体蛋白SjFz5在不同发育阶段的组织定位及mRNA表达水平分析

mRNA expression level and localization of the SjFz5,the receptor of Wnt signal pathway in Schistosoma japonicum

为研究日本血吸虫(Sj)Wnt信号分子受体SjFz5(Frizzled5)在Sj生长发育中的作用,本实验对其在不同发育阶段的mRNA转录水平及组织分布进行了检测,并采用定量PCR方法比较SjFz5基因在Sj不同发育阶段间的mRNA水平差异。以7 d童虫cDNA为模板,扩增SjFz5基因编码胞外区CRD(Cystein rich domain)的基因片段,构建pET-SjFz5-CRD表达重组质粒进行原核表达。以纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,并进行SjFz5蛋白的组织定位。结果显示:SjFz5基因mRNA在发育早期表达水平相对较高,其中7 d童虫中表达量最高,13 d和18 d童虫下调了约1/3。23 d及其后雌雄虫的表达水平继续下调,雄虫下调比雌虫略显缓慢。整个成虫阶段SjFz5 mRNA维持在一个相对较低的水平。免疫组化结果显示SjFz5蛋白在虫体组织中分布广泛,其中雌雄虫生殖器官组织中的分布最为明显。随着卵巢和睾丸的逐步发育,SjFz5蛋白的量也呈现增加的趋势。SjFz5 mRNA在7 d童虫内表达量最高,SjFz5蛋白在雌雄生殖器官组织中分布最为明显,提示其介导的Wnt信号通路可能参与调节童虫阶段的细胞增殖、器官分化,并可能调节两性生殖细胞的发育。

To investigate the potential function of SjFz5 （Schistosoma japnicum Frizzled5）, as a receptor of the Wnt signal pathway, during growth and development of Schistosome, mRNA expression levels of SjFz5 gene were analyzed at different developmental stage by real-time PCR. Extracellular CRD of SjFz5 gene was amplified from cDNA of 7 day schistosomulum by PCR, and inserted into pET28a for prokaryotic expression to prepare the polyclonal antibody against SjFzS. The distribution of SjFz5 proteins were detected by immunohistochemical staining with the prepared anti-SjFz5 polyclonal antibody. Real-time PCR analysis revealed that SjFz5 transcripts were highly expressed in the schistosomulum. The expression level of SjFz5 mRNA reached the highest at day 7, and then declined 1/3 at day 13 and day 18. In adult stages, SjFz5 mRNA maintained at low level after day 23, compared to schistosomulum stages. The immunohistochemical localization analysis showed that the SjFz5 protein broadly distributed in the musculature and acetabulum of schistosomulum and adult worms. Furthermore, SjFz5 was found prominently expressed in the tissues of the male and the ovary as well as the vitellarium of the female. Our data suggested that SjFz5 might be an important Wnt receptor with potential functions in regulating the cell differentiation and proliferation in schistosomulum, as well as the development of the reproductive organs of both sexes in adult worm.