The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this st...The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line. The SWP26 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1. The transfer vector pFastBac1-swp26-egfp was transformed into the bacterium Escherichia coli DHl0Bac/Bombyx mori nucleopolyhedrovirus (BmNPV) to construct the recombinant vBmswp26-egfp bacmid. The vBmswp26-egfp bacmid DNA was then used to transfect BmN cells to obtain the recombinant baculovirus. Western blotting analysis of total protein lysates in BmN cells infected by the recombinant virus showed a protein band of approximately 51 kDa, which corresponded to the deduced molecular weight of the swp26-egfp fusion protein. In addition, a fluorescence signal was observed in the cytoplasm and nucleoplasm of transfected cells, indicating that SWP26 had been successfully expressed in BmN cells. The SWP26 expression system established in this study lays the foundation for additional molecular and cellular studies, especially those focused on the interaction between the SWP26 protein of Nosema bombycis and the proteins of the silkworm, Bombyx mori.展开更多
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...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。展开更多
文摘The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line. The SWP26 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1. The transfer vector pFastBac1-swp26-egfp was transformed into the bacterium Escherichia coli DHl0Bac/Bombyx mori nucleopolyhedrovirus (BmNPV) to construct the recombinant vBmswp26-egfp bacmid. The vBmswp26-egfp bacmid DNA was then used to transfect BmN cells to obtain the recombinant baculovirus. Western blotting analysis of total protein lysates in BmN cells infected by the recombinant virus showed a protein band of approximately 51 kDa, which corresponded to the deduced molecular weight of the swp26-egfp fusion protein. In addition, a fluorescence signal was observed in the cytoplasm and nucleoplasm of transfected cells, indicating that SWP26 had been successfully expressed in BmN cells. The SWP26 expression system established in this study lays the foundation for additional molecular and cellular studies, especially those focused on the interaction between the SWP26 protein of Nosema bombycis and the proteins of the silkworm, Bombyx mori.
