本文選題：熱休克蛋白70　切入點：熱休克蛋白90　出處：《華中科技大學》2009年碩士論文　論文類型：學位論文

【摘要】：HSP70和HSP90在細胞內許多信號轉導途徑中起到十分重要的作用。研究表明在細胞應激時,HSP70或HSP90與蛋白激酶B(PKB)的結合對PKB的活性調節(jié)有至關重要的影響,但HSP70和HSP90與PKB之間如何發(fā)生相互作用的分子機制仍不清楚。 我們給予瞬時轉染PKB的HEK293T細胞短暫熱休克刺激,以PKB為誘餌蛋白運用PULL-DOWN技術檢測HSP90和/或HSP70與PKB的相互作用,并采用格爾德霉素(HSP90特異性抑制劑)處理上述細胞。結果發(fā)現(xiàn):HSP90和HSP70均與PKB發(fā)生相互作用,當格爾德霉素抑制HSP90與PKB的結合時,PKB與HSP70的相互作用顯著增強。由此我們推測:在HSP90被抑制的細胞內,細胞應激反應(如熱休克)可由HSP70與PKB的結合量增加而發(fā)生補償。但反之如果將HSP70與PKB的結合抑制后,PKB與HSP90的結合是否同樣可發(fā)生補償效應值得我們進一步研究。 由于目前缺乏理想的HSP70特異性抑制劑,我們將HSP70基因中的蛋白結合域(PBD)缺失,構建HSP70的兩種缺失突變體,其中一種只含HSP70的ATP域,另一種含HSP70-ATP域和CT域,使其失去與蛋白激酶B結合的功能。經雙酶切及DNA測序鑒定后,兩種真核表達重組體均成功構建,分別命名為truncated-ATP、truncated-ATP-CT。我們將這兩種真核表達重組體瞬時轉染HEK293T細胞,實驗分組為:(1)未轉染組;(2)全長HSP70轉染組;(3)truncated-ATP轉染組;(4)truncated-ATP-CT轉染組。WESTERN-BLOT檢測各組HSP70表達水平,結果表明:我們所構建的這兩種重組體均在HEK293T細胞內成功表達。這將為后期進一步闡明HSP70/HSP90與PKB的相互作用的分子機制奠定實驗基礎。
[Abstract]:HSP70 and HSP90 play an important role in many signal transduction pathways in cells. Studies have shown that the binding of HSP70 or HSP90 to protein kinase BPKB plays a crucial role in the regulation of PKB activity during cellular stress. However, the molecular mechanism of the interaction between HSP70 and HSP90 and PKB remains unclear. We gave transient heat shock stimulation to HEK293T cells transfected with PKB. PKB was used as bait protein to detect the interaction between HSP90 and / or HSP70 and PKB using PULL-DOWN technique. The cells were treated with the specific inhibitor of Gerd mycin HSP90). The results showed that both HSP70 and HSP70 interact with PKB. When Gerd mycin inhibited the binding of HSP90 to PKB, the interaction between HSP70 and PKB was significantly enhanced. The cellular stress response (such as heat shock) can be compensated by the increase of HSP70 / PKB binding, but on the contrary, if the binding between HSP70 and PKB is inhibited, the compensatory effect between HSP70 and HSP90 is worth further study. Due to the lack of ideal HSP70 specific inhibitors, we deleted the protein binding domain of HSP70 gene and constructed two deletion mutants of HSP70, one of which contains only ATP domain of HSP70, the other one contains HSP70-ATP domain and CT domain. After double enzyme digestion and DNA sequencing, two kinds of eukaryotic expression recombinant were successfully constructed, named truncated-ATP truncated-ATP-CT.These two eukaryotic expression recombinant were transiently transfected into HEK293T cells. The whole HSP70 transfection group was divided into 3 truncated-ATP transfection groups. The expression level of HSP70 was detected in the transfected group (.Western-BLOT). The results showed that the two recombinant proteins were successfully expressed in HEK293T cells, which would lay an experimental foundation for further elucidation of the molecular mechanism of the interaction between HSP70/HSP90 and PKB at a later stage.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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相關期刊論文 前1條

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本文編號：1622815