人热休克蛋白70功能域的基因克隆及其真核表达载体的构建

发布时间：2018-03-17 02:35

本文选题：热休克蛋白70　切入点：热休克蛋白90　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：HSP70和HSP90在细胞内许多信号转导途径中起到十分重要的作用。研究表明在细胞应激时,HSP70或HSP90与蛋白激酶B(PKB)的结合对PKB的活性调节有至关重要的影响,但HSP70和HSP90与PKB之间如何发生相互作用的分子机制仍不清楚。我们给予瞬时转染PKB的HEK293T细胞短暂热休克刺激,以PKB为诱饵蛋白运用PULL-DOWN技术检测HSP90和/或HSP70与PKB的相互作用,并采用格尔德霉素(HSP90特异性抑制剂)处理上述细胞。结果发现:HSP90和HSP70均与PKB发生相互作用,当格尔德霉素抑制HSP90与PKB的结合时,PKB与HSP70的相互作用显著增强。由此我们推测:在HSP90被抑制的细胞内,细胞应激反应(如热休克)可由HSP70与PKB的结合量增加而发生补偿。但反之如果将HSP70与PKB的结合抑制后,PKB与HSP90的结合是否同样可发生补偿效应值得我们进一步研究。由于目前缺乏理想的HSP70特异性抑制剂,我们将HSP70基因中的蛋白结合域(PBD)缺失,构建HSP70的两种缺失突变体,其中一种只含HSP70的ATP域,另一种含HSP70-ATP域和CT域,使其失去与蛋白激酶B结合的功能。经双酶切及DNA测序鉴定后,两种真核表达重组体均成功构建,分别命名为truncated-ATP、truncated-ATP-CT。我们将这两种真核表达重组体瞬时转染HEK293T细胞,实验分组为:(1)未转染组;(2)全长HSP70转染组;(3)truncated-ATP转染组;(4)truncated-ATP-CT转染组。WESTERN-BLOT检测各组HSP70表达水平,结果表明:我们所构建的这两种重组体均在HEK293T细胞内成功表达。这将为后期进一步阐明HSP70/HSP90与PKB的相互作用的分子机制奠定实验基础。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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