SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究
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摘要
目的鉴于纤维介素蛋白(fgl2)与严重急性呼吸综合征(severe acute respiratory syndrome, SARS)患者血栓形成有密切联系,本课题旨探索SARS冠状病毒结构蛋白与人纤维介素基因(hfgl2)的相互作用机制,为防治SARS提供新的靶点。
方法从SARS尸检病毒灭活肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、M和S2全长基因序列,再分别克隆到真核表达载体pcDNA3.1上。构建了SARS冠状病毒结构蛋白真核表达质粒pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2。分别与hfgl2启动子荧光素酶报告基因质粒及β半乳糖苷酶基因质粒(β-Gal )共转染到CHO细胞,采用免疫组织化学鉴定病毒蛋白的表达。通过检测报告基因荧光素酶(LUC)及β半乳糖苷酶的表达活性,反映病毒蛋白对hfgl2启动子的转录激活作用。将一系列hfgl2启动子区5’端缺失而保留共同3’端的启动子虫荧光素酶报告基因质粒,与SARS冠状病毒蛋白真核表达质粒共转染CHO细胞,并检测各组细胞的相对荧光素酶活性,明确在病毒蛋白刺激下该基因5’端非编码区对转录激活起重要作用的转录调控序列。
结果
1.通过酶切和核苷酸测序表明:目的片段成功克隆至pcDNA3.1,构建了SARS冠状病毒结构蛋白真核表达质粒pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1 -S2。免疫组织化学染色可见明显的CHO细胞胞浆棕染,说明N基因、M基因和S2基因的真核表达质粒构建成功,能够在蛋白水平表达。
2.通过与hfgl2启动子共转染实验表明:SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高6倍。
3.通过与一系列hfgl2启动子区段共转染实验表明:
转染前三个质粒hfgl2p(-1334)LUC、hfgl2p(-997)LUC、hfgl2p(-816)LUC的细胞荧光素酶活性改变无显著差异,但转染hfgl2p(-468)LUC质粒的细胞荧光素酶的活性较前显著降低,说明在hfgl2基因启动子-816位至-468位(相对于翻译起始点)之间存在着SARS冠状病毒N蛋白激活该基因的调控序列。结论
本研究成功构建SARS冠状病毒结构蛋白真核表达质粒,揭示了SARS冠状病毒N蛋白具有增强hfgl2基因转录活性的功能,明确了在SARS冠状病毒核心(N)蛋白作用下与该基因表达有关的调控序列,初步探讨了SARS-CoV与宿主基因间的相互作用,为进一步研究SARS冠状病毒的细胞信号传导途径奠定了基础。本研究的意义
近来研究表明SARS患者的肺组织中有血栓形成[5]。根据血栓形成区fgl2基因的高表达现象,我们推测hfgl2基因参与SARS的炎性病变。本研究分别构建了SARS冠状病毒结构蛋白N、M和S2的真核表达质粒和hfgl2启动子荧光素酶报告基因质粒(hfgl2p-LUC)。将两者共转染中国仓鼠卵巢(CHO)细胞,通过检测相对荧光素酶的活性,探讨SARS冠状病毒蛋白与宿主基因hfgl2的相互作用及其机制。将构建的一系列hfgl2启动子区段与SARS冠状病毒蛋白真核表达质粒共转染CHO细胞,并检测各组细胞的相对荧光素酶活性,探讨了在SARS冠状病毒核心(N)蛋白作用下与该基因表达有关的调控序列,也为下一步研究该基因表达有关的顺式作用元件及反式作用因子打下了基础。
Objective To investigate the responsible structural protein of SARS-associated coronavirus(SARS-CoV) in the activation of hfgl2 gene and to characterize the region in the hfgl2 promotor which is responsive to protein of SARS-CoV.
Methods SARS-CoV was isolated from patient with SARS and gene fragments encoding nucleocapsid protein, membrane protein and spike protein were obtained by RT-PCR. and they were sub-cloned into eukaryotic expression vector( pcDNA3.1).The orientation and the sequence were ensured by restriction endonucleases and sequencing assays. The recombinated plasmid was then transfected into Chinese hamster ovary (CHO) cells, and immunohistochemistry was employed to detect the expression of the structural proteins of SARS-CoV. To investigate the the responsible structural protein of SARS-CoV, Expression vectors of recombinant structural proteins N ,M and S2 of SARS-CoV were cotransfected respectively with hfgl2 promoter/luciferase reporter gene in CHO cells, and luciferase activity was detected for the assessment of promoter function.Constructs containing progressive deletions of the–1334 base pair fragment were cotransfected with N gene constrcts in CHO cells. Relative luciferase activity is expressed in fold increase compared with CHO cells cotransfected with the hfgl2 promoter constructs with PGL2-basic vector.
Results Expression vectors of recombinant structural proteins of SARS-CoV were successfully constructed. Immunohistochemistry showed that structural protein of SARS-CoV was expressed only in those cells transfected with the recombinated plasmid. Cotransfection of N gene expression construct with a reporter construct containing hfgl2 promoter in CHO cells showed a remarkable increasing in luciferase activity compared with nontransfected CHO cells. However, CHO cells cotransfected with pcDNA3.1-M and pcDNA3.1-S2 with hfgl2 promoter/luciferase reporter gene did not show significant difference in luciferase activity when compared with negative controls. Preliminary mapping of the hfgl2 promoter has defined a region from–816 to–468 to be responsive to induction of N protein.
Conclusions Nucleocapsid protein of SARS-CoV upregulates the transcription of hfgl2 prothrombinase/fibroleukin gene. The regulatory elements in the (–816 to–468) hfgl2 promoter account for the activation of hfgl2 gene in response to N protein of SARS-CoV.
方法从SARS尸检病毒灭活肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、M和S2全长基因序列,再分别克隆到真核表达载体pcDNA3.1上。构建了SARS冠状病毒结构蛋白真核表达质粒pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2。分别与hfgl2启动子荧光素酶报告基因质粒及β半乳糖苷酶基因质粒(β-Gal )共转染到CHO细胞,采用免疫组织化学鉴定病毒蛋白的表达。通过检测报告基因荧光素酶(LUC)及β半乳糖苷酶的表达活性,反映病毒蛋白对hfgl2启动子的转录激活作用。将一系列hfgl2启动子区5’端缺失而保留共同3’端的启动子虫荧光素酶报告基因质粒,与SARS冠状病毒蛋白真核表达质粒共转染CHO细胞,并检测各组细胞的相对荧光素酶活性,明确在病毒蛋白刺激下该基因5’端非编码区对转录激活起重要作用的转录调控序列。
结果
1.通过酶切和核苷酸测序表明:目的片段成功克隆至pcDNA3.1,构建了SARS冠状病毒结构蛋白真核表达质粒pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1 -S2。免疫组织化学染色可见明显的CHO细胞胞浆棕染,说明N基因、M基因和S2基因的真核表达质粒构建成功,能够在蛋白水平表达。
2.通过与hfgl2启动子共转染实验表明:SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高6倍。
3.通过与一系列hfgl2启动子区段共转染实验表明:
转染前三个质粒hfgl2p(-1334)LUC、hfgl2p(-997)LUC、hfgl2p(-816)LUC的细胞荧光素酶活性改变无显著差异,但转染hfgl2p(-468)LUC质粒的细胞荧光素酶的活性较前显著降低,说明在hfgl2基因启动子-816位至-468位(相对于翻译起始点)之间存在着SARS冠状病毒N蛋白激活该基因的调控序列。结论
本研究成功构建SARS冠状病毒结构蛋白真核表达质粒,揭示了SARS冠状病毒N蛋白具有增强hfgl2基因转录活性的功能,明确了在SARS冠状病毒核心(N)蛋白作用下与该基因表达有关的调控序列,初步探讨了SARS-CoV与宿主基因间的相互作用,为进一步研究SARS冠状病毒的细胞信号传导途径奠定了基础。本研究的意义
近来研究表明SARS患者的肺组织中有血栓形成[5]。根据血栓形成区fgl2基因的高表达现象,我们推测hfgl2基因参与SARS的炎性病变。本研究分别构建了SARS冠状病毒结构蛋白N、M和S2的真核表达质粒和hfgl2启动子荧光素酶报告基因质粒(hfgl2p-LUC)。将两者共转染中国仓鼠卵巢(CHO)细胞,通过检测相对荧光素酶的活性,探讨SARS冠状病毒蛋白与宿主基因hfgl2的相互作用及其机制。将构建的一系列hfgl2启动子区段与SARS冠状病毒蛋白真核表达质粒共转染CHO细胞,并检测各组细胞的相对荧光素酶活性,探讨了在SARS冠状病毒核心(N)蛋白作用下与该基因表达有关的调控序列,也为下一步研究该基因表达有关的顺式作用元件及反式作用因子打下了基础。
Objective To investigate the responsible structural protein of SARS-associated coronavirus(SARS-CoV) in the activation of hfgl2 gene and to characterize the region in the hfgl2 promotor which is responsive to protein of SARS-CoV.
Methods SARS-CoV was isolated from patient with SARS and gene fragments encoding nucleocapsid protein, membrane protein and spike protein were obtained by RT-PCR. and they were sub-cloned into eukaryotic expression vector( pcDNA3.1).The orientation and the sequence were ensured by restriction endonucleases and sequencing assays. The recombinated plasmid was then transfected into Chinese hamster ovary (CHO) cells, and immunohistochemistry was employed to detect the expression of the structural proteins of SARS-CoV. To investigate the the responsible structural protein of SARS-CoV, Expression vectors of recombinant structural proteins N ,M and S2 of SARS-CoV were cotransfected respectively with hfgl2 promoter/luciferase reporter gene in CHO cells, and luciferase activity was detected for the assessment of promoter function.Constructs containing progressive deletions of the–1334 base pair fragment were cotransfected with N gene constrcts in CHO cells. Relative luciferase activity is expressed in fold increase compared with CHO cells cotransfected with the hfgl2 promoter constructs with PGL2-basic vector.
Results Expression vectors of recombinant structural proteins of SARS-CoV were successfully constructed. Immunohistochemistry showed that structural protein of SARS-CoV was expressed only in those cells transfected with the recombinated plasmid. Cotransfection of N gene expression construct with a reporter construct containing hfgl2 promoter in CHO cells showed a remarkable increasing in luciferase activity compared with nontransfected CHO cells. However, CHO cells cotransfected with pcDNA3.1-M and pcDNA3.1-S2 with hfgl2 promoter/luciferase reporter gene did not show significant difference in luciferase activity when compared with negative controls. Preliminary mapping of the hfgl2 promoter has defined a region from–816 to–468 to be responsive to induction of N protein.
Conclusions Nucleocapsid protein of SARS-CoV upregulates the transcription of hfgl2 prothrombinase/fibroleukin gene. The regulatory elements in the (–816 to–468) hfgl2 promoter account for the activation of hfgl2 gene in response to N protein of SARS-CoV.
引文
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2. Hofmann H, Hattermann K, Marzi A, et al. S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients[J]. J Virol,2004,78(12):6134-42.
3. He Y, Zhou Y, Siddiqui P, et al. Identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus[J]. J Clin Microbiol,2005 ,43(8):3718-3726.
4. Li S, Lin L, Wang H, et al. The epitope study on the SARS-CoV nucleocapsid protein. Genomics Proteomics Bioinformatics[J], 2003 1(3): 198-206.
5. Liu S, Xiao G, Chen Y, et al. Interaction between heptad repeat 1 and
2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors[J]. Lancet, 2004,363(9413):938-947.
6. Fang X, Ye L, Timani KA, et al. Peptide domain involved in the interaction between membrane protein and nucleocapsid protein of SARS-associated coronavirus[J]. J Biochem Mol Biol, 2005,38(4):381-385.
7. Xu XJ, Gao XM. Immunological Responses against SARS-Coronavirus Infection in Humans[J]. Cell Mol Immunol, 2004,1(2):119-122.
8. Levy GA, Liu MF, Ding JW, et al. Molecular and functional analysis of the human prothrombinases gene(HFGL2) and its role in viral hepatitis[J]. Am J Pathology,2000,156:1217-1224.
9. Ding J W, Ning Q, Liu M F, et al. Fulminant hepatic failure in murinehepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase[J]. J Virol, 1997,71(12): 9223-9230.
10. Ning Q, Brown D, Parodo J, et al. Ribavirin inhibits viral-induced acrophage production of TNF, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response[J]. J Immunol, 1998,160(7): 3487-3493.
11. Marsden PA, Ning Q, Fung LS, et al. The fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosi in experimental and human viral hepatitis[J]. J Clin Invest, 2003,112(1):58-66.
12. SUN Yi, NING Qin, LI jinwen, et al . Expression of mgfl2/fibroleukin in mouse cardiac allograft and its potential clinical implication[J].Chin J transplant (中华器官移植杂志),2003,24(5):294-297.
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2. Hofmann H, Hattermann K, Marzi A, et al. S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients[J]. J Virol,2004,78(12):6134-42.
3. He Y, Zhou Y, Siddiqui P, et al. Identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus[J]. J Clin Microbiol,2005 ,43(8):3718-3726.
4. Li S, Lin L, Wang H, et al. The epitope study on the SARS-CoV nucleocapsid protein. Genomics Proteomics Bioinformatics[J], 2003 1(3): 198-206.
5. Liu S, Xiao G, Chen Y, et al. Interaction between heptad repeat 1 and
2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors[J]. Lancet, 2004,363(9413):938-947.
6. Fang X, Ye L, Timani KA, et al. Peptide domain involved in the interaction between membrane protein and nucleocapsid protein of SARS-associated coronavirus[J]. J Biochem Mol Biol, 2005,38(4):381-385.
7. Xu XJ, Gao XM. Immunological Responses against SARS-Coronavirus Infection in Humans[J]. Cell Mol Immunol, 2004,1(2):119-122.
8. Levy GA, Liu MF, Ding JW, et al. Molecular and functional analysis of the human prothrombinases gene(HFGL2) and its role in viral hepatitis[J]. Am J Pathology,2000,156:1217-1224.
9. Ding J W, Ning Q, Liu M F, et al. Fulminant hepatic failure in murinehepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase[J]. J Virol, 1997,71(12): 9223-9230.
10. Ning Q, Brown D, Parodo J, et al. Ribavirin inhibits viral-induced acrophage production of TNF, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response[J]. J Immunol, 1998,160(7): 3487-3493.
11. Marsden PA, Ning Q, Fung LS, et al. The fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosi in experimental and human viral hepatitis[J]. J Clin Invest, 2003,112(1):58-66.
12. SUN Yi, NING Qin, LI jinwen, et al . Expression of mgfl2/fibroleukin in mouse cardiac allograft and its potential clinical implication[J].Chin J transplant (中华器官移植杂志),2003,24(5):294-297.
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