微小隐孢子虫微线蛋白GP900与MDBK细胞互作蛋白的筛选

Screening of MDBK cell proteins interacted with Cryptosporidium parvum GP900

通过筛选与微小隐孢子虫微线蛋白GP900相互作用的MDBK细胞蛋白,为进一步揭示微小隐孢子虫入侵宿主细胞的分子机制提供依据。首先构建重组原核表达质粒pGEX-4T-GP900,表达并纯化重组GP900蛋白;然后将重组GP900蛋白与MDBK细胞裂解液孵育,免疫共沉淀获得结合蛋白,质谱法分析与GP900互作的蛋白;最后用酵母双杂交进行验证。结果表明,成功构建pGEX-4T-GP900表达质粒,并纯化获得大小为43 kDa的重组GP900蛋白;免疫沉淀-质谱法筛选到2种与GP900相互作用的MDBK细胞蛋白,分别为膜联蛋白A2和热休克蛋白5;酵母双杂交技术验证了GP900与以上2种蛋白均有相互作用。为进一步阐明微小隐孢子虫入侵宿主细胞的分子机制奠定了基础。

By screening bovine kidney cell MDBK proteins that interact with C.parvum microneme protein GP900,we provided the basis for further revealing the molecular mechanism of C.parvum invasion into host cells.First,the pGEX-4T-GP900 plasmid was constructed,and the recombinant protein GP900 was express and purified.Then the recombinant protein GP900 was incubated with MDBK cell lysates,and the binding proteins were screened by immunoprecipitation,the proteins interacting with GP900 were analyzed by mass spectrometry.Finally,the screened host proteins were verified by yeast two-hybrid technique.The results showed that the prokaryotic expression plasmid of pGEX-4T-GP900 was successfully constructed,and 43 kDa recombinant GP900 proteins were obtained.Two MDBK cell proteins interacting with GP900 were identified by immunoprecipitation-mass spectrometry,which were linked protein A2 and heat shock protein 5.The interaction between GP900 and these two proteins was verified by yeast two-hybrid technique.These results lay a foundation for studying the molecular mechanism of C.parvum invading host cells.