论文部分内容阅读
目的 寻找新的与霍乱毒素A1亚基 (CTA1)突变体 (S6 3F)相互作用的蛋白 ,来探讨CT佐剂活性的分子机理。方法 应用酵母双杂交技术 ,以CTA1(S6 3F)为诱饵蛋白筛选人脾细胞cDNA文库 ,并通过共转染、免疫共沉淀和Western杂交在哺乳动物细胞COS 7中确证诱饵蛋白和候选蛋白之间的相互作用。结果 筛选获得一个与HLAⅠ类分子α亚基高度同源的蛋白 ,这个蛋白在酵母核内以及COS 7细胞中都显示出与CTA1(S6 3F)的相互作用。结论 CTA1(S6 3F)与HLAⅠ类分子α亚基的相互作用可能导致HLAⅠ类分子进入“膜筏” ,引起膜筏不断聚集增大 ,聚集到一定程度后筏相关酪氨酸蛋白激酶活化 ,启动胞内信号的级联传递 ,增强免疫反应
OBJECTIVE: To search for new proteins that interact with cholera toxin A1 subunit (CTA1) mutant (S6 3F) to explore the molecular mechanism of CT adjuvant activity. Methods The cDNA library of human spleen cells was screened by CTA1 (S6 3F) as a bait protein by yeast two-hybrid technique. The expression of bait protein and protein candidate was verified by co-transfection, co-immunoprecipitation and Western blotting in mammalian cells COS 7 Interaction. As a result, a protein highly homologous to the α subunit of HLA class I molecule was screened, and this protein showed an interaction with CTA1 (S6 3F) both in yeast nucleus and in COS 7 cells. Conclusion The interaction between CTA1 (S6 3F) and α subunit of HLA class I molecules may lead to the entry of HLA class I molecules into “membrane rafts”, resulting in the continual accumulation of membrane rafts. After aggregation to a certain extent, the raft-associated tyrosine protein kinases activate and initiate Cascade transmission of intracellular signals to enhance the immune response