应用抑制性消减杂交技术筛选p7TP3剪切体1反式激活基因
发布时间：2018-10-13 08:34
【摘要】： 丙型肝炎病毒(HCV)的p7蛋白是存在于HCV结构蛋白和非结构蛋白之间的63个氨基酸残基组成的一个小蛋白,它在脂质膜中形成阳离子通道。p7蛋白对病毒颗粒感染性的产生是必需的。利用基因芯片技术对p7蛋白表达载体转染的肝母细胞瘤细胞系HepG2的基因表达谱变化进行了比较,发现p7蛋白可以反式调节某些基因的表达,其中包括一个未知功能基因,命名为p7TP3。在研究p7TP3基因的过程中,发现了p7TP3基因的不同剪切体,对p7TP3基因组进行分析,确定了p7TP3剪切体1的编码序列。为进一步探讨p7TP3剪切体1基因在HCV致病过程中的作用,我们进行了如下研究: 1.采用RCR技术克隆扩增p7TP3剪切体1,并以T-A克隆法,将p7TP3剪切体1基因片段连入载体pGEM-T,经EcoR I和BamH I双酶切鉴定以及测序分析,成功克隆了p7TP3剪切体1基因。 2.构建荧光表达质粒pEGFP-C1-p7TP3剪切体1,瞬时转染HepG2细胞,并以空载体pEGFP-C1作为平行对照,检测p7TP3剪切体1的细胞内定位。荧光显微镜下观察发现,荧光表达质粒pEGFP-C1-p7TP3剪切体1瞬时转染HepG2细胞后,发现其亚细胞定位于细胞浆中。 3.构建真核表达质粒pcDNA3.1/myc-his-p7TP3剪切体1 ,以表达质粒pcDNA3.1/myc-his-p7TP3剪切体1瞬时转染HepG2细胞,并以空载体pcDNA3.1/myc-his作为平行对照,制备转染后的细胞裂解液,应用抑制性消减杂交技术,构建p7TP3剪切体1反式激活相关基因差异表达的cDNA消减文库,筛选相关的靶基因片段,将产物与pGEM-Teasy载体连接,转染E. coli大肠杆菌系统进行文库扩增,随机挑选克隆,PCR扩增后进行测序及同源性分析,筛选得到14种上调基因。 结果表明:(1)p7TP3剪切体1蛋白具有反式调节功能。(2)筛选得到的cDNA全长序列,包括一些与细胞内信号转导、细胞生长调节、蛋白质翻译合成、物质代谢、细胞凋亡、免疫调节及肿瘤发生密切相关的蛋白编码基因,推测了p7TP3剪切体1在肝细胞内可能存在的调控机制。
[Abstract]:The p7 protein of hepatitis C virus (HCV) is a small protein consisting of 63 amino acid residues between HCV structural protein and nonstructural protein, which forms a cationic channel in lipid membrane. The gene expression profiles of hepatoblastoma cell line HepG2 transfected with p7 protein expression vector were compared by gene chip technique. It was found that p7 protein could transregulate the expression of some genes, including an unknown gene. Named p7TP3. In the course of studying the p7TP3 gene, different shearing bodies of p7TP3 gene were found. The p7TP3 genome was analyzed and the coding sequence of p7TP3 shearing 1 was determined. In order to further investigate the role of p7TP3 shearing 1 gene in the pathogenesis of HCV, we studied as follows: 1. RCR technique was used to clone and amplify p7TP3 splice 1, and T-A clone method was used to ligate the p7TP3 shearing 1 gene fragment into the vector pGEM-T, which was identified by EcoR I and BamH I double restriction endonuclease digestion and sequenced. P7TP3 shearing 1 gene was cloned successfully. The fluorescent expression plasmid pEGFP-C1-p7TP3 shunt 1 was constructed and transfected into HepG2 cells. The intracellular localization of p7TP3 shearing 1 was detected by using empty vector pEGFP-C1 as a parallel control. After transient transfection of fluorescent expression plasmid pEGFP-C1-p7TP3 shearing body 1 into HepG2 cells, it was found that the subcells were located in the cytoplasm of HepG2 cells. The eukaryotic expression plasmid pcDNA3.1/myc-his-p7TP3 splitter 1 was constructed, and the HepG2 cells were transiently transfected with the expression plasmid pcDNA3.1/myc-his-p7TP3 splitter 1. The transfected cell lysate was prepared using empty vector pcDNA3.1/myc-his as a parallel control, and the suppression subtractive hybridization technique was used. The cDNA subtractive library of p7TP3 shearing body 1 was constructed to transactivate differentially expressed genes, and the relevant target gene fragments were screened. The product was ligated with pGEM-Teasy vector and transfected into E. coli Escherichia coli system to amplify the library. The clones were randomly selected and sequenced and homologous analysis were performed after PCR amplification. Fourteen up-regulated genes were screened. The results showed that: (1) p7TP3 shearing body 1 protein had trans-regulation function. (2) the full-length sequence of cDNA was screened, including intracellular signal transduction, cell growth regulation, protein translation and synthesis, substance metabolism and apoptosis. The protein encoding genes closely related to immunomodulation and tumorigenesis inferred the possible regulatory mechanism of p7TP3 shearing 1 in hepatocytes.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2267960
[Abstract]:The p7 protein of hepatitis C virus (HCV) is a small protein consisting of 63 amino acid residues between HCV structural protein and nonstructural protein, which forms a cationic channel in lipid membrane. The gene expression profiles of hepatoblastoma cell line HepG2 transfected with p7 protein expression vector were compared by gene chip technique. It was found that p7 protein could transregulate the expression of some genes, including an unknown gene. Named p7TP3. In the course of studying the p7TP3 gene, different shearing bodies of p7TP3 gene were found. The p7TP3 genome was analyzed and the coding sequence of p7TP3 shearing 1 was determined. In order to further investigate the role of p7TP3 shearing 1 gene in the pathogenesis of HCV, we studied as follows: 1. RCR technique was used to clone and amplify p7TP3 splice 1, and T-A clone method was used to ligate the p7TP3 shearing 1 gene fragment into the vector pGEM-T, which was identified by EcoR I and BamH I double restriction endonuclease digestion and sequenced. P7TP3 shearing 1 gene was cloned successfully. The fluorescent expression plasmid pEGFP-C1-p7TP3 shunt 1 was constructed and transfected into HepG2 cells. The intracellular localization of p7TP3 shearing 1 was detected by using empty vector pEGFP-C1 as a parallel control. After transient transfection of fluorescent expression plasmid pEGFP-C1-p7TP3 shearing body 1 into HepG2 cells, it was found that the subcells were located in the cytoplasm of HepG2 cells. The eukaryotic expression plasmid pcDNA3.1/myc-his-p7TP3 splitter 1 was constructed, and the HepG2 cells were transiently transfected with the expression plasmid pcDNA3.1/myc-his-p7TP3 splitter 1. The transfected cell lysate was prepared using empty vector pcDNA3.1/myc-his as a parallel control, and the suppression subtractive hybridization technique was used. The cDNA subtractive library of p7TP3 shearing body 1 was constructed to transactivate differentially expressed genes, and the relevant target gene fragments were screened. The product was ligated with pGEM-Teasy vector and transfected into E. coli Escherichia coli system to amplify the library. The clones were randomly selected and sequenced and homologous analysis were performed after PCR amplification. Fourteen up-regulated genes were screened. The results showed that: (1) p7TP3 shearing body 1 protein had trans-regulation function. (2) the full-length sequence of cDNA was screened, including intracellular signal transduction, cell growth regulation, protein translation and synthesis, substance metabolism and apoptosis. The protein encoding genes closely related to immunomodulation and tumorigenesis inferred the possible regulatory mechanism of p7TP3 shearing 1 in hepatocytes.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
【共引文献】
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