Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

丙型肝炎病毒5'端非编码区结构域Ⅱ在其翻译启动活性中的作用

目的 分析丙型肝炎病毒(HCV)5'端非编码区(NCR)的结构域Ⅱ序列(nt44-118)在其翻译启动活性中的作用.方法 用PCR扩增技术获得缺失5'端118 nt的截短型HCV 5'NCR片段,并以之替换萤火虫荧光素酶(Flue)真核表达质粒pCMVNCRluc中的完整HCV 5'NCR,构建截短型HCV 5'NCR调控Fluc基因表达的真核表达质粒pCN1-d3.将pCN1-d3、pCMVNCRluc和pCMVNCRhc缺失HCV5'NCR nt1-43后的重组质粒pCN1-d2以脂质体方法 分别转染人肝癌细胞株HepG2,用双荧光素酶报告基因检测系统检测Fluc相对表达活性,RT-PCR检测Flue mRNA的相对表达水平.结果 酶切和测序结果 表明,重组质粒构建成功.各质粒转染细胞后Fluc mRNA的相对表达水平差异无统计学意义(P0.05);pCN1-d2表达的荧光素酶活性与pCMVNCRluc差异无统计学意义(P0.05),pCN1-d3的Fluc活性显著低于pCMVNCRIuc(P<0.01).结论 HCV 5'NCR的结构域Ⅱ(nt44-118)含有其发挥翻译启动功能的重要序列.     

HCV Core蛋白与IFN敏感性相关结构及功能的生物信息学分析

目的 生物信息学工具分析不同基因型HCV Core蛋白与IFN敏感性相关的结构功能及差异.方法 应用生物信息学工具对不同基因型HCV Core蛋白二级结构、三级结构、修饰位点及主要功能结构域预测分析.结果 HCV Core蛋白氨基酸序列、二级结构、三级结构各型间存在差异,可能存在酰胺化、cAMP依赖的激酶、PKC、TYR磷酸化等修饰位点及与两性蛋白SH3结构域、ERK、PKC相互作用的区域.结论 不同基因型HCV Core蛋白结构功能不同影响IFN治疗敏感性.     

HCV Core蛋白与IFN敏感性相关结构及功能的生物信息学分析

目的 生物信息学工具分析不同基因型HCV Core蛋白与IFN敏感性相关的结构功能及差异.方法 应用生物信息学工具对不同基因型HCV Core蛋白二级结构、三级结构、修饰位点及主要功能结构域预测分析.结果 HCV Core蛋白氨基酸序列、二级结构、三级结构各型间存在差异,可能存在酰胺化、cAMP依赖的激酶、PKC、TYR磷酸化等修饰位点及与两性蛋白SH3结构域、ERK、PKC相互作用的区域.结论 不同基因型HCV Core蛋白结构功能不同影响IFN治疗敏感性.     

HCV Core蛋白与IFN敏感性相关结构及功能的生物信息学分析

目的生物信息学工具分析不同基因型HCVCore蛋白与IFN敏感性相关的结构功能及差异。方法应用生物信息学工具对不同基因型HCVCore蛋白二级结构、三级结构、修饰位点及主要功能结构域预测分析。结果HCVCore蛋白氨基酸序列、二级结构、三级结构各型间存在差异,可能存在酰胺化、cAMP依赖的激酶、PKC、TYR磷酸化等修饰位点及与两性蛋白SH3结构域、ERK、PKC相互作用的区域。结论不同基因型HCVCore蛋白结构功能不同影响IFN治疗敏感性。     

丙型肝炎病毒非结构蛋白NS4B肝细胞结合蛋白基因的筛选与克隆

目的筛选并克隆人肝细胞cDNA文库中与丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)相互作用蛋白的基因,明确其具体作用机制。方法应用酵母双杂交系统3,将多聚酶链反应(PCR)法扩增的HCVNS4B基因连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和Xα半乳糖(Xαgal)上进行双重筛选阳性菌落,提取阳性酵母菌落的质粒转化大肠埃希菌,接种在氨苄西林LB平板上,选择生长菌落,提取质粒酶切鉴定,测序并在GenBank中进行生物信息学分析。结果成功克隆出HCVNS4B基因并在酵母细胞中表达,与肝文库配合后选出既能在4缺(SD/TrpLeuAdeHis)培养基又能在铺有Xαgal的4缺培养基上生长,并变成蓝色的真阳性菌落5个,序列分析显示,筛选到的肝细胞蛋白编码基因参与细胞代谢、生物氧化、生长调节等多种生物学过程。结论成功克隆出HCVNS4B蛋白与肝细胞相互作用蛋白,为进一步研究NS4B蛋白的功能,阐明HCV致病的分子生物学机制提供了新线索。     

HCV非结构蛋白质5A基因全长和高免疫源区的表达及免疫活性检测

目的探讨HCV非结构蛋白质5A（NS5A）基因全长和高免疫源区的表达并比较其检测灵敏度。方法以含HCV全基因组的质粒pBR^tm／HCV-3011（1b型）为模板，采用PCR方法扩增出NS5A全长基因（以下用NS5AL代替）和高免疫源区基因（以下用NS5AS代替），与pET-32a载体相连接构建重组表达载体pET-NS5AL和pET-NS5AS，分别转化E.coli Rosetta（DE3）pLysS和BL21（DE3）感受态细胞，经异丙幕-β-D-硫代半乳糖苷（IPTG）诱导后，蛋白质印迹法检测其表达，镍螯合（Ni2^＋-NTA）琼脂糖亲和层析柱纯化重组NS5AL和NS5AS蛋白，最后通过ELISA法检测重组蛋白的免疫活性并对其榆测灵敏度进行比较。结果SDS—PAGE鉴定与蛋白质印迹验证结果表明，重组NS5AL和NS5AS蛋白均得到很好地表达；纯化的重组NS5AL和NS5AS蛋白浓度分别为0．146和0．426mg／ml，纯度均〉96％；ELISA检测结果表明，重组NS5AL和NS5AS蛋白均具有较好的免疫活性，且重组NS5AL蛋白的检测灵敏度明显高于NS5AS。结论成功地表达出重组NS5AL和NS5AS蛋白，均具有较高的免疫活性，将有助于提高HCV临床检测的灵敏度。