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AY358935基因抗水疱口炎病毒的作用及其机制探讨

The Anti-virus Effect of AY358935 Gene on Vesicular Stomatitis Virus and the Mechanism Study

    摘要
  • 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)×105 PFU/mL、(6.25±2.05)×106 PFU/mL、(7.75±2.54)×106 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。

     

    Abstract:
      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  ① Virus 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)×105 PFU/mL, (6.25±2.05)×106 PFU/mL and (7.75±2.54)×106 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.

     

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