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目的干扰素调节因子(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新的剪接异构体,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)及逆转录聚合酶链反应,生物信息学方法比较新序列,亚克隆IRF-3c至pcDNA3.1-flag,转染人胚胎肾上皮293细胞,用抗flag抗体作Western blot分析,用萤虫素酶功能分析的方法观测IRF-3c对病毒诱导的干扰素β启动子萤虫素酶活性的影响。结果发现了一种新的IRF3剪接异构体IRF-3c,IRF-3c用了紧接第六外显子后的180个第六内含子的碱基,新引入的最初第2、3、4个新碱基为一终止密码子,导致IRF-3c蛋白多肽终止于第327位。Western blot出现预期的相对分子质量为44000的IRF-3c的蛋白强阳性条带。新的引用外显子序列可在小鼠的ETS表达库中找到同源序列,提示这种新的剪接体在生物演进中有其保守功能,IRF-3c萤虫素酶功能分析显示,该剪接体能抑制病毒诱导的干扰素β启动子活性至对照组的40%~50%。结论新的剪接体的发现为IRF-3这一重要分子的功能调节提供了新的线索,它是病毒感染通路中干扰素的显负性抑制剂,提示其功能为干扰素产生的精细调节成分,可防止干扰素的过度产生,其特异性序列可用于分析它们在人类疾病中的病理意义。
Objective Interferon regulatory factor (IRF) 3 is an important transcription factor that regulates the transcription of interferon gene after viral infection, looking for a new splicing isoform of IRF-3 and studying its structure and function. Methods RNA was extracted from human cells. The primers of IRF-3 published sequence were designed for rapid amplification of cDNA ends (RACE) and reverse transcriptase-polymerase chain reaction (PCR). Bioinformatics methods were used to compare the novel sequences. The IRF-3c was subcloned into pcDNA3.1 -flag, transfected human embryonic kidney 293 cells, with anti-flag antibody for Western blot analysis, the use of the function of the luciferase assay of IRF-3c on the virus-induced interferon β promoter luciferase activity . As a result, a novel IRF3 splice variant IRF-3c was found which used the base of the 180th sixth intron immediately after the sixth exon and the newly introduced first, second, Four new bases are a stop codon, leading to the termination of the IRF-3c polypeptide at position 327. Western blot showed a strong protein positive band for IRF-3c with a predicted relative molecular mass of 44000. The new reference exon sequences can find homologous sequences in the mouse ETS expression library, suggesting that this new spliceosome has its conserved function in the biological evolution. IRF-3c luciferase function analysis showed that the splicing Physiological inhibition of virus-induced interferon beta promoter activity to the control group of 40% to 50%. Conclusion The discovery of the new spliceosome provides new clues to the functional regulation of this important molecule of IRF-3, which is a negative inhibitor of interferon in the viral infection pathway, suggesting its function as a finely tuned component of interferon production , To prevent the overproduction of interferon, its specific sequence can be used to analyze their pathological significance in human disease.