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

【摘要】：HSP70和HSP90在細(xì)胞內(nèi)許多信號轉(zhuǎn)導(dǎo)途徑中起到十分重要的作用。研究表明在細(xì)胞應(yīng)激時,HSP70或HSP90與蛋白激酶B(PKB)的結(jié)合對PKB的活性調(diào)節(jié)有至關(guān)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(jī)制仍不清楚。 我們給予瞬時轉(zhuǎn)染PKB的HEK293T細(xì)胞短暫熱休克刺激,以PKB為誘餌蛋白運(yùn)用PULL-DOWN技術(shù)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(xì)胞。結(jié)果發(fā)現(xiàn):HSP90和HSP70均與PKB發(fā)生相互作用,當(dāng)格爾德霉素抑制HSP90與PKB的結(jié)合時,PKB與HSP70的相互作用顯著增強(qiáng)。由此我們推測:在HSP90被抑制的細(xì)胞內(nèi),細(xì)胞應(yīng)激反應(yīng)(如熱休克)可由HSP70與PKB的結(jié)合量增加而發(fā)生補(bǔ)償。但反之如果將HSP70與PKB的結(jié)合抑制后,PKB與HSP90的結(jié)合是否同樣可發(fā)生補(bǔ)償效應(yīng)值得我們進(jìn)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(jié)合域(PBD)缺失,構(gòu)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(jié)合的功能。經(jīng)雙酶切及DNA測序鑒定后,兩種真核表達(dá)重組體均成功構(gòu)建,分別命名為truncated-ATP、truncated-ATP-CT。我們將這兩種真核表達(dá)重組體瞬時轉(zhuǎn)染HEK293T細(xì)胞,實(shí)驗分組為:(1)未轉(zhuǎn)染組;(2)全長HSP70轉(zhuǎn)染組;(3)truncated-ATP轉(zhuǎn)染組;(4)truncated-ATP-CT轉(zhuǎn)染組。WESTERN-BLOT檢測各組HSP70表達(dá)水平,結(jié)果表明:我們所構(gòu)建的這兩種重組體均在HEK293T細(xì)胞內(nèi)成功表達(dá)。這將為后期進(jìn)一步闡明HSP70/HSP90與PKB的相互作用的分子機(jī)制奠定實(shí)驗基礎(chǔ)。
[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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R363

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相關(guān)期刊論文 前1條

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本文編號：1622815