虾肝肠胞虫孢壁蛋白SWP7的多克隆抗体制备和定位分析

Polyclonal antibody preparation and fluorescence localization analysis of Enterocytozoon hepatopenaei spore wall protein 7(SWP7)

孢壁作为微孢子虫最外层的结构,是连接微孢子虫与外界环境的桥梁。因此,研究孢壁蛋白对阐明宿主-微孢子虫相互作用的机制具有重要意义。本研究以虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)孢壁蛋白7(spore wall protein 7,SWP7)为研究对象,首先通过生物信息学分析其序列基本特征,然后合成该基因序列、构建原核表达载体、诱导蛋白表达并纯化,最后制备多克隆抗体。通过Western blotting验证该抗体的特异性后,利用间接免疫荧光技术观察和分析该蛋白在成熟孢子上的定位情况。结果表明,SWP7的N末端存在信号肽序列,且无跨膜域和GPI锚定位点,可能作为分泌蛋白发挥功能。同时,预测到SWP7有多个磷酸化修饰位点,可能参与肝肠胞虫信号转导、免疫应答、受体激活等功能。间接免疫荧光结果显示SWP7均匀地表达于成熟EHP孢壁上,且可能参与到萌发过程中。研究结果可为明确SWP7在肝肠胞虫入侵宿主中的功能及其机制提供理论基础。

As the outermost structure of microsporidia,the spore wall is the bridge for microsporidia to communicate with the external environment.Therefore,studying the spore wall composition may play a key role in elucidating the mechanism of host-parasite interaction.In this study,the spore wall protein(SWP7)of Enterocytozoon hepatopenaei(EHP)was studied.Firstly,the basic characteristics of the SWP7 sequence were analyzed by bioinformatics.Then the gene sequence was synthesized,the prokaryotic expression vector was constructed,and the induced recombinant protein was purified.Finally,the anti-SWP7 rabbit polyclonal antibody was prepared.After the specificity verification of the antibody by western blotting,the localization of the SWP7 protein on mature spores was observed and analyzed by indirect immunofluorescence.The results showed that there was a signal peptide sequence in the N-terminal of SWP7,and there was none of transmembrane domain and GPI anchor site,which indicated that SWP7 may display the function as a secretory protein.Meanwhile,it was predicted that SWP7 had multiple phosphorylation sites,which may paticipate in the signal transduction,immune response,receptor activation and other functions.The results of indirect immunofluorescence showed that SWP7 was uniformly expressed in the spore wall of mature EHP,which might participate in the germination process.The results lay a foundation for further study on the function and mechanism of SWP7 in the EHP invasion.