(精编)【医学教学课件】细胞因子课件教学课件.pptVIP

酵母双杂交系统的建立与发展 酵母转录子Gal4分子由一条多肽链组成，含有881个氨基酸。它有两个结构域： 1、 DNA结合结构域（DNA binding domain, DB ）由位于 N-末端1~147个氨基酸构成，能识别位于Gal4基因的上游激活序列（UAS, upstream activating sequence）,此外， 在其N-端还具有一段核定位序列； 2、转录激活结构域（activation domain, AD）由位于C-末端的768~881位氨基酸构成。 当Gal4 的两个结构域位于不同肽链上，只要它们在空间上充分接近，则能够恢复Gal4作为转录因子的活性. Normal Transcription. Normal transcription requires both the DNA-binding domain (BD) and the activation domain (AD) of a transcriptional activator (TA). (DB) (AD) Reporter gene a reporter gene, which is simply a gene whose protein product can be easily detected and measured. X Y Ga14的DB Ga14的AD 诱饵(bait) 猎物(prey) 报道基因 表达 报道基因的基因产物 通过对报道基因表达产物的检测，反过来可判别作为“诱饵”和“猎物”的两个蛋白之间是否可存在相互作用。 Plasmid construction. The bait and hunter fusion proteins are constructed in the same manner. The bait DNA is isolated and inserted into a plasmid adjacent to the GAL4 DB DNA. When this DNA is transcribed, the bait protein will now contain the GAL4 DNA-binding domain as well. The ‘prey fusion protein contains the GAL4 AD. ?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? The yeast two-hybrid assay uses two plasmid constructs: the bait plasmid, which is the protein of interest fused to a GAL4 binding domain, and the hunter plasmid, which is the potential binding partner fused to a GAL4 activation domain. Ga14蛋白 调控的GAL1 序列 ?-半乳糖苷酶 的LacZ Field改造LacZ基因，将其整合到酵母菌染色体URA3位上 URA3 URA3 酵母菌的GaL4基因和GaL80基因（ Gal80是Gal4的负调控基因）被缺失 报道基因 IL-2、 IL-4、 IL-5 IL-4 + IL-5 IL-4 IFN-? 5. Antagonism 3. Redundancy 4.synergy Stimulating the proliferation of the B cells More effectively inducing the exchange of IgE IL-4 interdicts the action that IFN- ? induces Ig exchanges B B B _ + On stimulation, rapid synthesis , release , action and degradation. 6. Rapidity 7. high potency Infinitesimal cytokines perform remarkable biological effects（pM,10-12 M). Perform in the non-specific manner, and not restric