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目的 干扰素调节因子 (interferonregulatoryfactors ,IRF) 3是病毒感染后调节干扰素基因转录重要的转录因子 ,寻找IRF 3新的基因拼接异构体 ,研究其结构及功能。方法 抽提人类细胞RNA ,用IRF 3已发表序列设计引物 ,作cDNA末端快速放大 (RACE)及RT PCR ,生物信息学方法比较新序列 ,亚克隆IRF 3b至 pcDNA3.1 flag ,转染人胚胎肾上皮 2 93细胞 ,用抗flag抗体作Westernblot分析 ,用荧光素酶功能分析的方法观测IRF 3b对病毒诱导的干扰素 β启动子荧光素酶活性的影响。结果 发现了一种新的IRF 3拼接异构体IRF 3b ,IRF 3b用了紧接第七外显子前的 1 6个第六内含子的碱基 ,导致阅读框架移位 ,相应蛋白质的C端第 32 8～ 4 52位为不同于IRF 3的新C末端。Westernblot出现预期的相对分子质量 (Mr)为 57.75× 1 0 3的IRF 3b蛋白强阳性条带。新的外显子序列可在小鼠的表达序列标签 (EST)表达库中找到同源序列 ,提示这种新的异构体在生物演进中有其保守功能 ,IRF 3b荧光素酶功能分析显示 ,该同分异构体能抑制病毒诱导的干扰素 β启动子活性至对照组的 4 0 %～ 50 %。结论 新的异构体的发现为IRF 3这一重要分子的功能调节提供了新的线索 ,它可能是病毒感染通路中干扰素的显性负性抑制剂 ,提示其功能为干扰素产生的?
OBJECTIVE: Interferon regulatory factor (IRF) 3 is an important transcription factor that regulates the transcription of interferon gene after viral infection, and searches for new splicing isoforms of IRF 3 to study its structure and function. Methods RNA was extracted from human cells. The primers were designed according to the published sequence of IRF 3. RACE and RT PCR were used to amplify the cDNA. The sequences of IRF 3b to pcDNA3.1 were subcloned by bioinformatics methods. Renal epithelial 293 cells were analyzed by Western blot with anti-flag antibody. The effect of IRF 3b on the luciferase activity of interferon beta promoter was observed by luciferase assay. As a result, a novel IRF 3 spliced isoform IRF 3b was found, which used 16 sixth intron bases immediately before the seventh exon, resulting in a shift in the reading frame of the corresponding protein C-terminal 32 8 ~ 4 52 is different from the new C-terminal IRF 3. Westernblot showed a strong positive band of IRF 3b protein with the expected relative molecular mass (Mr) of 57.75 × 10 3. The new exon sequence can find homologous sequences in mouse EST expression library, suggesting that this new isoform has its conserved function in biological evolution. The functional analysis of IRF 3b luciferase , The isomer can inhibit the virus-induced interferon β promoter activity to 40% ~ 50% of the control group. Conclusion The discovery of new isomers provides new clues to the functional regulation of IRF 3, an important molecule that may be a dominant negative inhibitor of interferon in the viral infection pathway, suggesting its function as an interferon.