丙型肝炎病毒p7蛋白反式调节作用的研究
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摘要
目的丙型肝炎病毒(HCV)是慢性肝炎的主要病原之一,目前世界上约有1.7 亿人被感染,常导致严重肝病,包括肝硬化和肝细胞癌(HCC)。HCV的基因组含有一个长的开放读码框(ORF),编码一个约3010 个氨基酸残基(aa)组成的多蛋白,该多蛋白被细胞和病毒蛋白酶切割产生至少10 个病毒基因产物。p7 蛋白是其中之一,在脂质膜中形成六聚体阳离子通道,金刚烷胺和长烷基链的亚氨基糖衍生物可抑制p7 形成的阳离子通道。p7 对HCV病毒颗粒的感染产生是必需的,但关于其功能研究知之甚少,本研究旨在探讨p7 蛋白在HCV 致病过程中的作用。
方法
1. 构建p7 的真核表达载体pcDNA3.1(-)-p7,将其与pCAT3-Promoter 共转染人肝癌细胞系HepG2,48 h 后收获细胞,用酶联免疫吸附法(ELISA)检测细胞中氯霉素乙酰转移酶(CAT)的表达活性。
2. 以表达质粒pcDNA3.1(-)-p7 瞬时转染HepG2 细胞,并以空载体pcDNA3.1(-)做为平行对照,制备转染后的细胞裂解液。应用抑制性消减杂交(SSH) 技术,构建p7 反式调节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T/A 载体连接,构建cDNA 消减文库,并转化大肠杆菌进行文库扩增,随机挑选克隆,PCR 扩增后进行测序及同源性分析。对转染后的细胞裂解液同时应用基因表达谱芯片技术对差异表达mRNA进行检测和分析。
3. 构建去唾液酸糖蛋白受体1(ASGPR1)启动子的报告基因载体pCAT3-ASGPR1p,并以其转染肝癌HepG2 细胞系,用ELISA 法检测CAT的表达活性,并与pcDNA3.1(-)-p7 共转染HepG2 细胞系,用ELISA 法检测CAT 的表达活性。
结果
1. pCAT3-promoter 和pcDNA3.1(-)-p7 共转染的HepG2 细胞的CAT 表达活性较pCAT3-promoter 下降了87%。
2. 成功构建人HCV p7 蛋白反式调节相关基因差异表达的cDNA 文库,包括15 个上调基因和6 个下调基因。我们提取高质量的总RNA 并进行逆转
Objective Hepatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma. HCV infects an estimated 170 million persons worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. HCV p7 is one of these protein. Recombinant HCV p7 could form ion channels in artificial lipid membranes. The ion transport observed in lipid membranes could be blocked by a known ion channel inhibitor, amantadine. HCV p7 is essential for infectivity of HCV. Its actual role in the virus life cycle has not been determined. We design such an experiment to explicit the molecular biological mechanisms of p7 after HCV infection.
Methods 1.The recombined expression plasmid pcDNA3.1(-)-p7 was constructed, HepG2 cells were cotransfected with pcDNA3.1(-)-p7 and pCAT3-Promoter. After 48 h, cells were collected and detected for the expression of the chloramphenicol acetyltransferase (CAT) by an enzyme-linked immunosorbent assay (ELISA) kit. 2.HepG2 cells were transiently transfected with pcDNA3.1(-)-p7 , and pcDNA3.1(-) empty vector was used as control. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-p7 and pcDNA3.1(-) empty vector,respectively,and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained production was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5αin random. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. At the same time, Microarray was employed for detecting and analyzing of both mRNA from the HepG2 cells. 3. We construct the expression plasmid pCAT3-ASGPR1p, which was transfected into the hepatoblastoma cell line HepG2, then cotransfected with pcDNA3.1(-)-p7. The activity of CAT in HepG2 cells transfected was detected by a ELISA kit after 48
hours.
Results 1.The activity of CAT in HepG2 cells transfected by the pcDNA3.1(-)-p7 was 87 percent lower than that of HepG2 cells transfected by control plasmid. 2.The subtractive library of genes transregulated by HCVp7 was constructed successfully. From the SSH results,it was found 15 genes were up-regulated and 6 genes were down-regulated by p7 protein of HCV. High quality mRNA and cDNA had been prepared and successful microarray screening had been conducted. From the scanning results, it was found 1 genes were up-regulated and 22 genes were down-regulated by p7. 3.The report vector pCAT3-ASGPR1p has been constructed and had been confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells co-transfected with pCAT3-ASGPR1p and pcDNA3.1(-)-p7 was 7.7 times as high as that of pCAT3-Basic, and 2.4 as high as that of pCAT3-ASGPR1p.
Conclusions The p7 protein of HCV has transregulatinging effect on SV40 promoter. The obtained sequences may be target genes transregulated by p7, among which some genes coding proteins involved in cell signal transduction, cycle regulation, translation and synthesis of protein, metabolism,apoptosis, immunoregulation, and correlated with tumor. p7 can transactivate ASGPR1 promoter, and so can up-regulate the expression of ASGPR1 gene. These may explain the possible mechanism of p7 in vivo.
方法
1. 构建p7 的真核表达载体pcDNA3.1(-)-p7,将其与pCAT3-Promoter 共转染人肝癌细胞系HepG2,48 h 后收获细胞,用酶联免疫吸附法(ELISA)检测细胞中氯霉素乙酰转移酶(CAT)的表达活性。
2. 以表达质粒pcDNA3.1(-)-p7 瞬时转染HepG2 细胞,并以空载体pcDNA3.1(-)做为平行对照,制备转染后的细胞裂解液。应用抑制性消减杂交(SSH) 技术,构建p7 反式调节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T/A 载体连接,构建cDNA 消减文库,并转化大肠杆菌进行文库扩增,随机挑选克隆,PCR 扩增后进行测序及同源性分析。对转染后的细胞裂解液同时应用基因表达谱芯片技术对差异表达mRNA进行检测和分析。
3. 构建去唾液酸糖蛋白受体1(ASGPR1)启动子的报告基因载体pCAT3-ASGPR1p,并以其转染肝癌HepG2 细胞系,用ELISA 法检测CAT的表达活性,并与pcDNA3.1(-)-p7 共转染HepG2 细胞系,用ELISA 法检测CAT 的表达活性。
结果
1. pCAT3-promoter 和pcDNA3.1(-)-p7 共转染的HepG2 细胞的CAT 表达活性较pCAT3-promoter 下降了87%。
2. 成功构建人HCV p7 蛋白反式调节相关基因差异表达的cDNA 文库,包括15 个上调基因和6 个下调基因。我们提取高质量的总RNA 并进行逆转
Objective Hepatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma. HCV infects an estimated 170 million persons worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. HCV p7 is one of these protein. Recombinant HCV p7 could form ion channels in artificial lipid membranes. The ion transport observed in lipid membranes could be blocked by a known ion channel inhibitor, amantadine. HCV p7 is essential for infectivity of HCV. Its actual role in the virus life cycle has not been determined. We design such an experiment to explicit the molecular biological mechanisms of p7 after HCV infection.
Methods 1.The recombined expression plasmid pcDNA3.1(-)-p7 was constructed, HepG2 cells were cotransfected with pcDNA3.1(-)-p7 and pCAT3-Promoter. After 48 h, cells were collected and detected for the expression of the chloramphenicol acetyltransferase (CAT) by an enzyme-linked immunosorbent assay (ELISA) kit. 2.HepG2 cells were transiently transfected with pcDNA3.1(-)-p7 , and pcDNA3.1(-) empty vector was used as control. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-p7 and pcDNA3.1(-) empty vector,respectively,and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained production was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5αin random. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. At the same time, Microarray was employed for detecting and analyzing of both mRNA from the HepG2 cells. 3. We construct the expression plasmid pCAT3-ASGPR1p, which was transfected into the hepatoblastoma cell line HepG2, then cotransfected with pcDNA3.1(-)-p7. The activity of CAT in HepG2 cells transfected was detected by a ELISA kit after 48
hours.
Results 1.The activity of CAT in HepG2 cells transfected by the pcDNA3.1(-)-p7 was 87 percent lower than that of HepG2 cells transfected by control plasmid. 2.The subtractive library of genes transregulated by HCVp7 was constructed successfully. From the SSH results,it was found 15 genes were up-regulated and 6 genes were down-regulated by p7 protein of HCV. High quality mRNA and cDNA had been prepared and successful microarray screening had been conducted. From the scanning results, it was found 1 genes were up-regulated and 22 genes were down-regulated by p7. 3.The report vector pCAT3-ASGPR1p has been constructed and had been confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells co-transfected with pCAT3-ASGPR1p and pcDNA3.1(-)-p7 was 7.7 times as high as that of pCAT3-Basic, and 2.4 as high as that of pCAT3-ASGPR1p.
Conclusions The p7 protein of HCV has transregulatinging effect on SV40 promoter. The obtained sequences may be target genes transregulated by p7, among which some genes coding proteins involved in cell signal transduction, cycle regulation, translation and synthesis of protein, metabolism,apoptosis, immunoregulation, and correlated with tumor. p7 can transactivate ASGPR1 promoter, and so can up-regulate the expression of ASGPR1 gene. These may explain the possible mechanism of p7 in vivo.
引文
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[2] Griffin SD, Beales LP, Clarke DS, et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett,2003,535(1-3):34~38
[3] Pavlovic D, Neville DC, Argaud O, et al. The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain minosugar derivatives.Proc Natl Acad Sci USA,2003,100(10):6104~6108
[4] Harada T, Tautz N, Thiel HJ. E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol, 2000,74(20):9498~ 9506
[5] Sakai A, Claire MS, Faulk K, et al. The p7 polypeptide of hepatitis C virus is critical for infectivity andcontains functionally important genotype-specific sequences. Proc Natl Acad Sci USA, 2003,100(20):11646~11651
[6] Lohmann V, Korner F, Koch J, et al.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science,1999,285:110~113
[7] Lin C, Lindenbach BD, Pragai BM, et al. Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol ,1994,68(5424):5063~5073
[8] Siebert PD, Chenchik A, Kellogg DE, et al. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res,1995,23 (6):1087~ 1091
[9] 成军,李克,陆荫英,等. 丙型肝炎病毒调节基因区结合的研究进展. 世界华人消化杂志,2002,10(2):223~225
[10] 成军. 慢性病毒性肝炎发病机制的分子生物学研究. 世界华人消化杂志,2002,10(2):125~128
[11] 成军. 慢性丙型病毒性肝炎肝脏脂肪变的机制及其意义. 世界华人消化 杂志,2002,10(9):999~1003
[12] 刘妍,杨倩,成军,等. 基因表达谱芯片筛选NS5ATP3 转染细胞差异表达基因. 世界华人消化杂志,2004,12(2):306~310
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