AY358935基因抗水疱口炎病毒的作用及其机制探讨
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摘要
目的初步探讨本课题组前期克隆的AY358935基因对水疱口炎病毒(vesicular stomatitis virus,VSV)的作用及机制。方法将构建完整的pcDNA3.1-AY358935质粒及pcDNA3.1空载体分别稳定转染HEK293细胞,并以VSV感染已转染上述基因或空白的HEK293细胞,感染复数(multiplicity of infection,MOI)为0.001。蚀斑分析法检测以上3组细胞上清液中不同时间点的病毒滴度,感染VSV 24 h后用台盼蓝排斥试验检测各组细胞死亡率;对稳定转染目的基因的细胞提取总RNA,进行全基因组cDNA芯片分析。结果①病毒滴度:感染VSV 12 h后,pcDNA3.1-AY358935组细胞上清病毒滴度较pcDNA3.1组和空白组低。18 h时3组病毒滴度分别为(7.16±2.33)×10~5 PFU/mL、(6.25±2.05)×10~6 PFU/mL、(7.75±2.54)×10~6 PFU/mL,pcDNA3.1-AY358935组细胞上清病毒滴度与其余两组的差异接近10倍(P<0.01);②细胞死亡率:病毒感染24 h后,pcDNA3.1-AY358935组、pcDNA3.1组和空白组的细胞死亡率分别为(35.00±6.68)%、(78.33±15.03)%和(83.34±14.98)%,pcDNA3.1-AY358935组的细胞死亡率较其余两组降低(P<0.01);③基因芯片分析结果:与pcDNA3.1空载体组比较,稳定转染pcDNA3.1-AY358935的细胞有30个基因表达上调在3倍以上,其中,干扰素激活基因、干扰素效应基因、细胞因子与趋化因子所占比例分别为27%、17%、20%。结论 AY358935基因具有抗VSV作用,其机制可能涉及干扰素相关的天然免疫应答。
Objective To explore the anti-virus effect of AY358935 gene cloned by our research team on vesicular stomatitis virus(VSV), and studytheanti-virus mechanism. Methods HEK293 cells were stably transfected by the AY358935 gene recombinant plasmid pcDNA3.1-AY358935 or pcDNA3.1 blank plasmid respectively. Then VSV was added into the cell wells to infect the above cells at the multiplicity of infection(MOI) of 0.001. The virus titers in the liquid supernatant of the above three groups of cells were detected on different time,and the mortality of cells of each group was tested with trypan blue exclusion test at 24 h post VSV infection. Total RNA was extracted from the cells that stably transfected with target gene for the whole genome-wide cDNA microarray analysis. Results ①Virus titer:The virus titer in the liquid supernatant of pcDNA-3.1-AY358935 transfection cells group was obviously lower than those in pcDNA-3.1 transfection cell group and blank control cell group at 12 h post infection. The virus titerin the liquid supernatant of three groups were(7.16±2.33)×10~5 PFU/mL,(6.25±2.05)×10~6 PFU/mL and(7.75±2.54)×10~6 PFU/mL respectively at 18 h post infection. At that time, the virus titerin the liquid supernatant of pcDNA3.1-AY358935 group was nearly 10 times lower than those of other two groups(P<0.01). ②Mortality of cells:The cell mortality of pcDNA3.1-AY358935 group,pcDNA3.1 group and blank group were(35.00±6.68)%,(78.33±15.03)% and(83.34±14.98)% respectively at 24 h post infection.The cell mortality of pcDNA3.1-AY358935 group was significantly decreased comparing with other two groups(P<0.01). ③Result of genes chip analysis: compared with pcDNA3.1 group,30 cell genes were up-regulated by more than 3 times in pcDNA3.1-AY358935 group. Among them, the proportion of interferon-activating gene, interferon-effect gene, cytokine and chemokine was 27%, 17%, and 20%, respectively. Conclusion AY358935 gene hasan anti-VSV effect, and its anti-virus mechanism may involve the interferon-associated natural immune response.
Objective To explore the anti-virus effect of AY358935 gene cloned by our research team on vesicular stomatitis virus(VSV), and studytheanti-virus mechanism. Methods HEK293 cells were stably transfected by the AY358935 gene recombinant plasmid pcDNA3.1-AY358935 or pcDNA3.1 blank plasmid respectively. Then VSV was added into the cell wells to infect the above cells at the multiplicity of infection(MOI) of 0.001. The virus titers in the liquid supernatant of the above three groups of cells were detected on different time,and the mortality of cells of each group was tested with trypan blue exclusion test at 24 h post VSV infection. Total RNA was extracted from the cells that stably transfected with target gene for the whole genome-wide cDNA microarray analysis. Results ①Virus titer:The virus titer in the liquid supernatant of pcDNA-3.1-AY358935 transfection cells group was obviously lower than those in pcDNA-3.1 transfection cell group and blank control cell group at 12 h post infection. The virus titerin the liquid supernatant of three groups were(7.16±2.33)×10~5 PFU/mL,(6.25±2.05)×10~6 PFU/mL and(7.75±2.54)×10~6 PFU/mL respectively at 18 h post infection. At that time, the virus titerin the liquid supernatant of pcDNA3.1-AY358935 group was nearly 10 times lower than those of other two groups(P<0.01). ②Mortality of cells:The cell mortality of pcDNA3.1-AY358935 group,pcDNA3.1 group and blank group were(35.00±6.68)%,(78.33±15.03)% and(83.34±14.98)% respectively at 24 h post infection.The cell mortality of pcDNA3.1-AY358935 group was significantly decreased comparing with other two groups(P<0.01). ③Result of genes chip analysis: compared with pcDNA3.1 group,30 cell genes were up-regulated by more than 3 times in pcDNA3.1-AY358935 group. Among them, the proportion of interferon-activating gene, interferon-effect gene, cytokine and chemokine was 27%, 17%, and 20%, respectively. Conclusion AY358935 gene hasan anti-VSV effect, and its anti-virus mechanism may involve the interferon-associated natural immune response.
引文
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[24] DE VEER MJ,HOLKO M,FREVEL M,et al.Functional classification of interferon-stimulated genes identified using microarrays.J Leukoc Biol,2001,69(6):912-920.
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[2] 周丹,刘雪茹,李涛,等.小G蛋白Rab5对表达在HEK293 细胞上的大电导钙激活钾通道的影响.山东医药,2017,57(8):21-24.
[3] 孟越,谢苗苗,林振,等.甲状旁腺素受体真核表达载体的构建体稳定转染HEK293细胞系的建立.南方医科大学学报,2013,33(7):956-961.
[4] 蔡鑫泽,顾卉,刘彤,等.P16 hLMO1编码基因重组质粒的构建及蛋白的表达和定位.中国医科大学学报,2009,38(11):816-819.
[5] LI XY,KUANG Y,HUANG XJ,et al.Preparation and characterization of a new monoclonal antibody against CXCR4 using lentivirus vector.Int Immunopharmacol,2016,36:100-105.
[6] 薛贻敏,林风辉,刘艳丽,等.白细胞介素17A对柯萨奇B3病毒性心肌炎小鼠病毒复制的影响及其机制.临床心血管病杂志,2017,33(7):688-693.
[7] 程仕彤,冯鹭,刘丽娜,等.干扰素对肝纤维化小鼠肝组织中Foxp3 调节性T细胞的影响.中国医科大学学报,2013,42(5):402-405.
[8] GNANADURAI CW,FU ZF.CXCL10 and blood-brain barrier modulation in rabies virus infection.J Oncotarget,2016,7 (10):10694-10695.
[9] ZHAO A,LU W,DE LE.Functional synergism of human defensin 5 and human defensin 6.Biochem Biophys Res Commun,2015,467(4):967-972.
[10] CURRIE SM,FINDLAY EG,MCFARLANE AJ,et al.Cathelicidins have direct antiviral activity against respiratory syncytial virus in vitro and protective function in vivo in mice and humans.J Immunol,2016,196(6):2699-2710.
[11] PEEL E,CHENG Y,DJORDJEVIC JT,et al.Cathelicidins in the Tasmanian devil (Sarcophilus harrisii).Sci Rep,2016,6:35019.
[12] REBHANDL S,HUEMER M,GREIL R,et al.AID/APOBEC deaminases and cancer.Oncoscience,2015,2(4):320-333.
[13] LEI Q,GAO B,ZHANG D.Design and preparation of matrine surface-imprinted material and studies on its molecule recognition selectivity.J Biomater Sci Polym Ed,2016,27(1):1-21.
[14] SAFA A,RASHIDINEJAD HR,KHALILI M,et al.Higher circulating levels of chemokines CXCL10,CCL20 and CCL22 in patients with ischemic heart disease.Cytokine,2016,83:147-157[2018-10-10].https://doi.org/10.1016/j.cyto.2015.04.006.
[15] GRAWENHOFF J,ENGELMAN AN.Retroviral integrase protein and intasome nucleoprotein complex structures.World J Biol Chem,2017,8(1):32-44.
[16] YIN Y,LIU W,DAI Y.SOCS3 and its role in nassociated diseaseas.Hum Immunol,2015,76(10):775-780.
[17] CHOW KT,GALE MJ.SnapShot:interferon signaling.Cell,2015,163(7):1808e1[2018-10-10].https://doi.org/10.1016/j.cell.2015.12.008.
[18] 宪庆,伍参荣.2′-5′寡聚腺苷酸合成酶(OAS)的活化及抗病毒作用应用研究进展.中外健康文摘,2012,9(15):84-86.
[19] HUANG Y,HUANG X,CAI J,et al.Identification of orange-spotted grouper (Epinephelus coioides) interferon regulatory factor 3 involved in antiviral immune response against fish RNA virus.Fish Shellfish Immunol,2015,42 (2):345-352.
[20] HENKES LE,PRU JK,ASHLEY RL,et al.Embryo mortality in Isg15-/-mice is exacerbated by environmental stress.Biol Reprod,2015,92(2):36[2018-10-10].https://doi.org/10.1095/biolreprod.114.122002.
[21] DONNELLY RP,KOTENKO SV.Interferon-lambda:a new addition to an old family.J Interferon Cytokine Res,2010,30(8):555-564.
[22] GIBBERT K,SCHLAAKJ F,YANG D.IFN-alpha subtypes:distinct biological activities in anti-viral therapy.Br J Pharmacol,2013,168(5):1048-1058.
[23] DONNELLY RP,KOTENKO SV.Interferon-lambda:a new addition to an old family.J Interferon Cytokine Res,2010,30(8):555-564.
[24] DE VEER MJ,HOLKO M,FREVEL M,et al.Functional classification of interferon-stimulated genes identified using microarrays.J Leukoc Biol,2001,69(6):912-920.
[25] SCHNEIDER WM,CHEVILLOTTE MD,RICE CM.Interferon- stimulated genes:a complex web of host defenses.Annu Rev Immunol,2014,32:513-545[2018-10-10].https://doi.org/10.1146/annurev-immunol-032713-120231.
[26] CHEN Z,MARK C,TIEN YH,et al.Interferon-induced ISG15 pathway:an ongoing virus-host battle.Trends Microbiol,2013,21(4):181-186.