稳定过表达人源SAMHD1基因细胞株的建立及其抗病毒活性的研究
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摘要
目的建立慢病毒介导的稳定过表达人源SAMHD1基因的结肠癌细胞株,观察SAMHD1对细胞抗1型单纯疱疹病毒(HSV-1)感染能力的影响。方法利用pCDH-EF1-MCS-T2A-copGFP慢病毒载体新构建了p CDH-EF1-MCS-SAMHD1-T2A-GFP重组质粒,测序鉴定成功后,将重组质粒和慢病毒包装质粒共转染293T细胞,收集病毒液,浓缩后感染人结肠癌细胞,构建过表达细胞株。利用HSV-1感染过表达细胞株并与对照组比较,通过Western blotting和免疫荧光检测病毒感染效率。结果基因测序结果显示成功构建了重组质粒;Western blotting检测稳定过表达人源SAMHD1结肠癌细胞株构建成功;Western blotting和免疫荧光结果显示过表达人源SAMHD1的细胞株能够有效抑制HSV-1病毒感染和复制。结论成功构建慢病毒介导的稳定过表达人源SAMHD1基因细胞株,过表达人源SAMHD1的细胞株能有效抑制HSV-1病毒复制,为进一步深入研究SAMHD1蛋白生物学功能提供保障。
Objective A lentivirus-mediated colon cancer cell line stably overexpressing human SAMHD1 gene was constructed to observe the effect of this gene on the ability of the cells to resist herpes simplex virus type 1(HSV-1) infection. Methods p CDH-EF1-MCS-SAMHD1-T2 A-GFP recombinant plasmid was constructed using pCDH-EF1-MCS-T2 A-copGFP lentiviral vector. After confirming successful synthesis with sequencing,the recombinant plasmid and lentivirus packaging plasmids were co-transfected into 293 T cells. The pseudovirus solution was collected and concentrated,then human colon cancer cells were infected with the concentrated solution. The cell line overexpressing SAMHD1 gene was infected with HSV-1 in contrast to the control group,and the virus infection efficiency was assessed by Western blotting and immunofluorescence analyses. Results The pCDH-EF1-MCS-SAMHD1-T2 A-GFP recombinant plasmid was successfully constructed and verified by gene sequencing. Western blotting confirmed the overexpression of the SAMHD1 gene with higher levels of the gene in the transfected cells in contrast to control human colon cancer cell line. Western blotting and immunofluorescence showed that the cell line overexpressing human SAMHD1 gene could effectively inhibit the infection of HSV-1. Conclusion Lentivirus-mediated stable cell line overexpressing human SAMHD1 gene was effective in inhibiting HSV-1 replication and could help us to further investigate the function of SAMHD1.
Objective A lentivirus-mediated colon cancer cell line stably overexpressing human SAMHD1 gene was constructed to observe the effect of this gene on the ability of the cells to resist herpes simplex virus type 1(HSV-1) infection. Methods p CDH-EF1-MCS-SAMHD1-T2 A-GFP recombinant plasmid was constructed using pCDH-EF1-MCS-T2 A-copGFP lentiviral vector. After confirming successful synthesis with sequencing,the recombinant plasmid and lentivirus packaging plasmids were co-transfected into 293 T cells. The pseudovirus solution was collected and concentrated,then human colon cancer cells were infected with the concentrated solution. The cell line overexpressing SAMHD1 gene was infected with HSV-1 in contrast to the control group,and the virus infection efficiency was assessed by Western blotting and immunofluorescence analyses. Results The pCDH-EF1-MCS-SAMHD1-T2 A-GFP recombinant plasmid was successfully constructed and verified by gene sequencing. Western blotting confirmed the overexpression of the SAMHD1 gene with higher levels of the gene in the transfected cells in contrast to control human colon cancer cell line. Western blotting and immunofluorescence showed that the cell line overexpressing human SAMHD1 gene could effectively inhibit the infection of HSV-1. Conclusion Lentivirus-mediated stable cell line overexpressing human SAMHD1 gene was effective in inhibiting HSV-1 replication and could help us to further investigate the function of SAMHD1.
引文
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[13]JAUREGUI P,LANDAU NR.DNA damage induces a SAM-HD1-mediated block to the infection of macrophages by HIV-1[J].Sci Rep,2018,8(1):4153.DOI:10.1038/s41598-018-22432-4.
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[15]LI M,ZHANG D,ZHU M,et al.Roles of SAMHD1 in antiviral defense,autoimmunity and cancer[J].Rev Med Virol,2017,27(4):e1931.DOI:10.1002/rmv.1931.
[16]LEE EJ,SEO JH,PARK JH,et al.SAMHD1 acetylation enhances its deoxynucleotide triphosphohydrolase activity and promotes cancer cell proliferation[J].Oncotarget,2017,8(40):68517-68529.DOI:10.18632/oncotarget.19704.
[17]ST GC,WU L.SAMHD1:a new insight into HIV-1 restriction in myeloid cells[J].Retrovirology,2011,8(1):55.DOI:10.1186/1742-4690-8-55.
[18]ST GC,DE SILVA S,AMIE SM,et al.SAMHD1 restricts HIV-1infection in dendritic cells(DCs)by dNTP depletion,but its expression in DCs and primary CD4+T-lymphocytes cannot be upregulated by interferons[J].Retrovirology,2012,9(1):105.DOI:10.1186/1742-4690-9-105.
[19]GRAMBERG T,KAHLE T,BLOCH N,et al.Restriction of diverse retroviruses by SAMHD1[J].Retrovirology,2013,10:26.DOI:10.1186/1742-4690-10-26.
[20]徐兴丽,李琦涵.1型单纯疱疹病毒疫苗的研究进展[J].中国生物制品学杂志,2018,31(4):445-448.DOI:10.13200/j.cnki.cjb.002161.
[2]黄俊,仇超,张晓燕.SAMHD1抗艾滋病病毒作用研究进展[J].病毒学报,2012,28(4):477-481.DOI:10.13242/j.cnki.bingduxuebao.002307.
[3]DRAGIN L,NGUYEN LA,LAHOUASSA H,et al.Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply[J].Retrovirology,2013,10(1):30.DOI:10.1186/1742-4690-10-30.
[4]AMIE SM,NOBLE E,KIM B.Intracellular nucleotide levels and the control of retroviral infections[J].Virology,2013,436(2):247-254.DOI:10.1016/j.virol.2012.11.010.
[5]LAHOUASSA H,DADDACHA W,HOFMANN H,et al.SAMHD1restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates[J].Nat Immunol,2012,13(3):223-228.DOI:10.1038/ni.2236.
[6]RYOO J,HWANG SY,CHOI J,et al.SAMHD1,the Aicardi-Goutieres syndrome gene and retroviral restriction factor,is a phosphorolytic ribonuclease rather than a hydrolytic ribonuclease[J].Biochem Biophys Res Commun,2016,477(4):977-981.DOI:10.1016/j.bbrc.2016.07.013.
[7]CHEN S,BONIFATI S,QIN Z,et al.SAMHD1 suppression of antiviral immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways[J].Proc Natl Acad Sci USA,2018,115(16):E3798-E3807.DOI:10.1073/pnas.1801213115.
[8]ST GC,KIM SH,MAKSIMOVA VV,et al.A cyclin-binding motif in human SAMHD1 is required for its HIV-1 restriction,dNTPase activity,tetramer formation,and efficient phosphorylation[J].J Virol,2018,92(6):e01787-17.DOI:10.1128/JVI.01787-17.
[9]DU MOULIN M,NURNBERG P,CROW YJ,et al.Cerebral vasculopathy is a common feature in Aicardi-Goutieres syndrome associated with SAMHD1 mutations[J].Proc Natl Acad Sci USA,2011,108(26):E232,E233.DOI:10.1073/pnas.1104699108.
[10]RICE GI,BOND J,ASIPU A,et al.Mutations involved in aicardi-goutieres syndrome implicate SAMHD1 as regulator of the innate immune response[J].Nat Genet,2009,41(7):829-832.DOI:10.1038/ng.373.
[11]KYEI GB,CHENG X,RAMANI R,et al.Cyclin L2 is a critical HIVdependency factor in macrophages that controls SAMHD1 abundance[J].Cell Host Microbe,2015,17(1):98-106.DOI:10.1016/j.chom.2014.11.009.
[12]KRETSCHMER S,WOLF C,KONIG N,et al.SAMHD1 prevents autoimmunity by maintaining genome stability[J].Ann Rheum Dis,2015,74(3):e17.DOI:10.1136/annrheumdis-2013-204845.
[13]JAUREGUI P,LANDAU NR.DNA damage induces a SAM-HD1-mediated block to the infection of macrophages by HIV-1[J].Sci Rep,2018,8(1):4153.DOI:10.1038/s41598-018-22432-4.
[14]DADDACHA W,KOYEN AE,BASTIEN AJ,et al.SAMHD1 promotes DNA end resection to facilitate DNA repair by homologous recombination[J].Cell Rep,2017,20(8):1921-1935.DOI:10.1016/j.celrep.2017.08.008.
[15]LI M,ZHANG D,ZHU M,et al.Roles of SAMHD1 in antiviral defense,autoimmunity and cancer[J].Rev Med Virol,2017,27(4):e1931.DOI:10.1002/rmv.1931.
[16]LEE EJ,SEO JH,PARK JH,et al.SAMHD1 acetylation enhances its deoxynucleotide triphosphohydrolase activity and promotes cancer cell proliferation[J].Oncotarget,2017,8(40):68517-68529.DOI:10.18632/oncotarget.19704.
[17]ST GC,WU L.SAMHD1:a new insight into HIV-1 restriction in myeloid cells[J].Retrovirology,2011,8(1):55.DOI:10.1186/1742-4690-8-55.
[18]ST GC,DE SILVA S,AMIE SM,et al.SAMHD1 restricts HIV-1infection in dendritic cells(DCs)by dNTP depletion,but its expression in DCs and primary CD4+T-lymphocytes cannot be upregulated by interferons[J].Retrovirology,2012,9(1):105.DOI:10.1186/1742-4690-9-105.
[19]GRAMBERG T,KAHLE T,BLOCH N,et al.Restriction of diverse retroviruses by SAMHD1[J].Retrovirology,2013,10:26.DOI:10.1186/1742-4690-10-26.
[20]徐兴丽,李琦涵.1型单纯疱疹病毒疫苗的研究进展[J].中国生物制品学杂志,2018,31(4):445-448.DOI:10.13200/j.cnki.cjb.002161.