應用抑制性消減雜交技術篩選p7TP3剪切體1反式激活基因
發(fā)布時間：2018-10-13 08:34
【摘要】： 丙型肝炎病毒(HCV)的p7蛋白是存在于HCV結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白之間的63個氨基酸殘基組成的一個小蛋白,它在脂質(zhì)膜中形成陽離子通道。p7蛋白對病毒顆粒感染性的產(chǎn)生是必需的。利用基因芯片技術對p7蛋白表達載體轉(zhuǎn)染的肝母細胞瘤細胞系HepG2的基因表達譜變化進行了比較,發(fā)現(xiàn)p7蛋白可以反式調(diào)節(jié)某些基因的表達,其中包括一個未知功能基因,命名為p7TP3。在研究p7TP3基因的過程中,發(fā)現(xiàn)了p7TP3基因的不同剪切體,對p7TP3基因組進行分析,確定了p7TP3剪切體1的編碼序列。為進一步探討p7TP3剪切體1基因在HCV致病過程中的作用,我們進行了如下研究: 1.采用RCR技術克隆擴增p7TP3剪切體1,并以T-A克隆法,將p7TP3剪切體1基因片段連入載體pGEM-T,經(jīng)EcoR I和BamH I雙酶切鑒定以及測序分析,成功克隆了p7TP3剪切體1基因。 2.構(gòu)建熒光表達質(zhì)粒pEGFP-C1-p7TP3剪切體1,瞬時轉(zhuǎn)染HepG2細胞,并以空載體pEGFP-C1作為平行對照,檢測p7TP3剪切體1的細胞內(nèi)定位。熒光顯微鏡下觀察發(fā)現(xiàn),熒光表達質(zhì)粒pEGFP-C1-p7TP3剪切體1瞬時轉(zhuǎn)染HepG2細胞后,發(fā)現(xiàn)其亞細胞定位于細胞漿中。 3.構(gòu)建真核表達質(zhì)粒pcDNA3.1/myc-his-p7TP3剪切體1 ,以表達質(zhì)粒pcDNA3.1/myc-his-p7TP3剪切體1瞬時轉(zhuǎn)染HepG2細胞,并以空載體pcDNA3.1/myc-his作為平行對照,制備轉(zhuǎn)染后的細胞裂解液,應用抑制性消減雜交技術,構(gòu)建p7TP3剪切體1反式激活相關基因差異表達的cDNA消減文庫,篩選相關的靶基因片段,將產(chǎn)物與pGEM-Teasy載體連接,轉(zhuǎn)染E. coli大腸桿菌系統(tǒng)進行文庫擴增,隨機挑選克隆,PCR擴增后進行測序及同源性分析,篩選得到14種上調(diào)基因。 結(jié)果表明:(1)p7TP3剪切體1蛋白具有反式調(diào)節(jié)功能。(2)篩選得到的cDNA全長序列,包括一些與細胞內(nèi)信號轉(zhuǎn)導、細胞生長調(diào)節(jié)、蛋白質(zhì)翻譯合成、物質(zhì)代謝、細胞凋亡、免疫調(diào)節(jié)及腫瘤發(fā)生密切相關的蛋白編碼基因,推測了p7TP3剪切體1在肝細胞內(nèi)可能存在的調(diào)控機制。
[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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346
本文編號：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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346
【共引文獻】
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