人TANK结合激酶1的基因克隆、表达及生物活性鉴定

摘要

目的:克隆人TANK结合激酶l(TBK1)基因,构建其真核表达载体,检测该基因在293细胞中的表达,并利用萤光素酶报告基因实验检测其生物活性.方法:应用RT-PCR方法,以HeLa细胞RNA为模板,扩增获得TBK1基因,定向克隆到pcDNA3-Flag载体中,以LipofectAMINE2000转染试剂转染pcDNA-Flag-TBK1至293细胞中进行瞬时表达,并利用萤光素酶报告基因实验检测诱导β干扰素(IFN-β)转录的情况.结果:测序结果表明,从人HeLa细胞总RNA中克隆到正确的TBK1基因全长编码序列,利用Western印迹检测其在293细胞中获得有效表达,利用萤光素酶报告基因实验检测TBK1可以诱导IFN-β转录激活.结论:真核表达的人TBK1具有相应的生物学活性,为研究其功能奠定了基础.%Objective: To clone human TANK binding kinase-1 (TBK1) gene, express it in 293 cell lines and I-dentify its biological activities. Methods: TBK1 gene was amplified from RNA of HeLa cell lines by RT-PCR, and then inserted into eukaryotic expression vector pcDNA3-Flag. The recombinant expression vector was transiently transfected into 293 cell lines by LipofectAMINE2000 reagent. The function on activating interferon-f} (IFN-P) transcription was analyzed by luciferase reporter gene. Results: The sequence of the cloned TBK1 was confirmed to be correct by DNA sequencing, the result of immunoblotting showed that TBK1 was successfully expressed in 293 cell lines. Luciferase assay showed that the IFN-fi can significantly activated by TBK1. Conclusion: The human TBK1 gene was cloned and expressed in 293 cell lines, IFN-|3 significantly activated by TBK1, which provide us platform to purse TBK1 function in innate immunity.