SH_2结构域是最早鉴定的调节信号转导蛋白相互作用的蛋白质组件,它可以特异性识别磷酸化酪氨酸短序列,介导受体酪氨酸蛋白激酶信号转导途径.含有SH_2组件的一类蛋白质组成了SH_2信号蛋白家族,一般可分为两大类:一类是除了SH_2组件外还具有催化结构域的酶蛋白,如PI-3K,PLC-γ,Src;而另一类是无催化活性的接头蛋白,这类蛋白含有其它组件,和细胞内信号蛋白连接起来,如Grb2.近年来,又有一类SH_2信号蛋白被发现,这类蛋白唯有SH_2结构域,无其它任何蛋白质组件,通过竞争其它SH_2信号蛋白的结合位点,阻止下游信号募集,行使控制信号传导的负调控因子的功能.SH_2结构域是细胞内信号传导的重要组件,主要参与酪氨酸蛋白激酶相关受体介导的信号转导过程,是上游受体与下游信号分子联系的分子基础.人染色体8p22区是一个富含疾病基因的区域,是癌基因和抑癌基因的研究热点.在前期工作中,运用外显子捕获和外显子拼接等策略,我们在这一区域克隆了SH_2A基因,是SH_2信号蛋白家族的新成员,并构建了SH_2A基因的真核重组表达载体pcDNA3.1-SH_2A和pEGFP-SH_2A,通过细胞转染、激酶比活性检测、亚细胞定位、表达分析和流式细胞仪等方法对其功能进行了初步的研究.在多种肿瘤癌组织及癌旁组织中表达差异提示,它与肿瘤发生发展有一定的相关性.除此之外,对SH_2A基因的确切功能尚无报道.为了进一步明确SH_2A基因表达产物在细胞信号转导途径中的功能,我们构建了pcDNA3.1-SH_2A之结构域缺失型pcDNA3.1-SH_2A-Del及pcDNA3.1-SH_2A之SH_2结构域点突变型pcDNA3.1-SH_2A-Mut,试图通过这两种方法使SH_2结构域丧失功能,通过细胞转染、激酶比活性测定及尝试构建稳定表达细胞株等手段从另一个角度证明SH_2A基因在PKC信号转导途径中的抑制性作用.
    IntroducionThe Src homology 2 (SH_2) domain is a protein domain of about 100 amino - acid residues first identified as a conserved sequence region between the onco-proteins Src and Fps. Similar sequences were later found in many other intracel-lular signal - transducing proteins. SH_2 domains function as regulatory modules of intracellular signalling cascades by interacting with high affinity to phosphoty-rosine - containing target peptides in a sequence - specific and strictly phospho-rylation - dependent manner. SH_2 family plays an important role in signal trans-duction by tyrosine protein kinase. The expressional difference between tumor and tumor neighboring tissue suggested SH_2A might relate to tumor onset or development. 8p22 of human chromosome is a region rich in disease - related genes and is also the susceptible loci of oncogenes and tumor suppressor genes. In the previous work, we cloned SH_2A gene in this region by exon trapping and exon linking, which is a novel member of SH_2 family. Also,  we constructed its recombinant expression vector, and investigated its function by cell transfection, kinase assay, subcellular localization and expression analysis. SH_2 domain is considered as "Protein Recognized Code". Besides these, the exact function of SH_2 A gene has not been reported yet. So we constructed the SH_2 - domain - deleted type, named with pcDNA3.1 - SH2A - Del, and the SH_2 - domain - mu-tagenic type, named with pcDNA3.1 - SH_2A - Mut, and made a further investigation on its function by cell transfection, kinase assay, and expression analysis. And then we tried to create the stable cell lines using neomycin (Geneticin).Materials1. Bel7402 cell line and C0S7 cell line;2. Molecular Biology Reagents of Gene Cloning;3. Reagents for Kinase Assay;4. Cell Transfection Kit.MethodsBy SOE - PCR method to amplify the coding sequence of SH_2 A gene including the mutagenic points, eukaryotic recombined expression vector, pcDNA3. 1- SH_2 A - Mut was constructed; in the manner of digestion by EcoR and self -ligation by T_4 ligase of the pcDNA3. 1 - SH_2A, the most part of the SH_2 domain was knocked out of the plasmid and pcDNA3. 1 - SH_2A - Del was constructed. Kinase assay was performed to examine PKC activity in the transfected cells, Bel7402 & COS7, by the method of CaPO_4 - DNA coprecipitation. After linearizing the plasmids, the COS7 cell line was transfected by lipofection, and neomy-cin (Geneticin ) was expected to use to create stable cell lines.ResultsThe coding sequence of SH_2A gene, a fragment of 896bp was got by SOE -PCR, which contains mutagenic point. Eukaryotic recombined expression vector, pcDNA3.1 -SH_2A — Mut was constructed. Eukaryotic recombined expression vector, pcDNA3. 1 -SH_2A -Del was constructed by means of enzyme cutting and self - ligation. After transfection, in both cell lines, Bel7402 & COS7, expressing SH_2A gene, the cytoplasm PKC activity decreased 27. 3% and 27. 8% compared with the control groups, but still no apparent alteration was found in membrane PKC activity. We have expected to create stable cell lines, COS7- SH_2 A as well as the mutagenic and the domain - deleted types. After using

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