【作者】 周颖；

【导师】 李丰；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2005， 硕士

【摘要】 目的 PAK1(P21-activated kinase1)是一保守的丝氨酸/苏氨酸蛋白激酶,为Rho家族小鸟苷三磷酸酶(Rho-GTPase)Cdc42和Rac下游重要的靶蛋白,参与许多重要的细胞活动。PAK1主要分布在脑、肌肉、脾中。PAK1包含N-端调节区和C-末端激酶区,它的N-末端调节区含GTP酶结合域(GTPase binding domain,GBD),它可介导PAK1与Rac和Cdc42的结合。研究表明,PAK1具有广泛的生物学功能,包括细胞骨架的动力学调节,细胞移动,生存和凋亡,细胞周期,基因转录调节,细胞生长信号转导和转化等,而这些功能的完成是由于PAK1磷酸化其下游靶蛋白引起的。到目前为止,已经发现了多种PAK1的底物及其相互作用蛋白。并且许多证据足以表明在肿瘤发生发展过程中存在PAK1信号转导途径。本研究通过观察PAK1重组蛋白在原核细胞中的表达情况及PAK1在真核细胞中的定位,为进一步研究PAK1的生物学性能提供理论和实验基础。 方法 1.总RNA提取及cDNA合成: 总RNA提取按照RNA提取protocol操作完成;在M-MuLV Reverse Transcriptase作用下逆转录为cDNA。 2.以cDNA为模板扩增PAK1 依据PAK1序列,设计2对引物,建立PCR反应体系,进行PCR循环设计,扩增PAK1。 3.重组质粒的构建 将PAK1基因克隆到GST融合蛋白原核表达载体pGEX-5X-1中,构建重组质粒pGEX-5X-1/PAK1;同时,利用基因重组技术将PAK1基因更多还原

【Abstract】 ObjectivePAK1 ( P21 - activated kinase 1) is an evolutionarily conserved serine/threonine kinases, are becoming increasingly important for a variety of cellular functions in mammalian cells. Pak1 acts as downstream effectors for the small GTPas-es, Cdc42 and Racl. Pak1 is highly expressed in the brain, muscle, and spleen. PAK1 contains an N - terminal regulatory domain and a C - terminal kinase domain, its N -terminal regulatory domain contains GTPase binding domain to mediate the binding of PAK1 to Rac/Cdc42. Accumulating evidence indicates that PAK1 is important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, cell cycle and angiogenesis. Pak1 are involved in the regulation of cellular function via phosphorylating a number of downstream target protein. Many evidences showed that Pak1 activation occurs during the process of tumorigenesis, and Pak1 is likely to provide insight into the role of Paks in human cancers. In this study,I detect PAK1 expression in pro-karyocyte and its location in transfected eukaiyocyte so as to provide basis for further study of PAK1.MethodsTotal RNA was isolated using Tripure agent. The cDNA of all kinds of tissues were prepared using M - MuLV Reverse Transcriptase and Oligo dT primer.Techniques of gene recombination and gene transfection were employed.The coding sequences of PAK1 were cloned into the procaryotic expression vector pGEX - 5X - 1 , generating the recombinant plasmids pGEX - 5X - 1/ PAK1, expressed in BL21 after transformation and IPTG induction. In the first stage of a Western Blot, SDS -PAGE electrophoresis was used to separate proteins, and the proteins was seperated on the gel based on size. In the second stage of the Western Blot, Proteins from the gel were transferred onto a membrane using electrophoresis. The proteins was transferred to the membrane in the same position as they are on the gel. Finally, I used anti - GST SRP and PAK1 antibodies to detect the GST -PAK1 fusion protein.At the same time,PAK1 cDNA was ligated to EGFP gene of pEGFP - C1. The recombinant plasmid was transfected into cancer cell line with liposome, and the expression of GFP - PAK1 fusion protein was observed by fluorescent microscopy .ResultsAfter isolation of total RNA, the samples were quantitated on the basis of absorbance at 260/280nm. Their concentration and 260/280 value could meet the need of its application.When the recombinant plasmid was detected with double endonulease analysis , two fragments could be released from it. And their size is correspondent to vector and target gene respectively. Moreover, sequence analysis indicated that PAK coding sequence was completely correct. These evidences showed that PAK1 recombinant plasmid was successfully constructed.GST - PAK1 fusion protein could be expressed in 1 、2、4 hrs after IPTG induction and reached its peak at 2hr. Western blot analysis indicated that the fusion protein could specifically bind to anti - GST and PAK1 antibody. So we may determine that PAK1 recombinant protein could be expressed in prokaryo-cyte.Under fluorescent microscopy, cells which highly expressed GFP protein showed green fluorescence. The expression of PAK1 was observed in cytoplasm transfected with pEGFP - PAK1.更多还原