TBK1相关激酶活性缺失突变体和泛素样结构域突变体的构建、表达及生物活性鉴定

摘要

Objective: To construct the TANK binding kinase-l(TBKl) kinasedead mutant and ubiquitin-like domain mutant eukaryotic expressing plasmid, express them in 293 cell lines and identify theirs biological activities. Methods: According to the reported TBK1 mutant sequence and QuickChange Site-Directed Mutagenesis kit, two of specific DNA sequences were designed and synthesized. Using the plasmid of TBK1 which was constructed by researchers in our laboratory as templates, construct TBK1 kinasedead mutant and ubiquitin-like domain mutant eukaryotic expressing plasmid, named pcDNA3-Flag-TBKl (KD) and pcDNA3-Flag-TBKl (AULD). The recombinant expression vector pcDNA-Flag-TBKl was transiently transfected into 293 cell lines by LipofactAMINE2000 reagent. The function on activating interferon-P(IFN-p) transcription was analyzed by luciferase reporter gene. Results: The sequences of the TBK1 kinasedead mutant and ubiquitin-like domain mutant eukaryotic expressing plasmid were confirmed to be correct by DNA sequencing, the result of immunoblotting showed that TBK1 kinasedead mutant and ubiquitin-like domain mutant were successfully expressed in 293 cell lines. The activity of inducing IFN-ji transcription by transfected TBK1 kinasedead mutant and ubiquitin-like domain mutant was significantly lower thanwild type TBK1. Conclusion: The TBK1 kinasedead mutant and ubiquitin-like domain mutant expressing plasmid was constructed, and successfully expressed in 293 cell lines, IFN-3 induction of these mutant are significantly lower than wild type TBK1, which lays the foundation for further investigating the role of TBK1.%目的:构建TANK结合激酶1(TBK1)相关激酶活性缺失突变体和泛素样结构域突变体真核表达载体,检测该基因相关突变体在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:根据文献报道的突变序列及QuickChange Site-Directed Mutagenesis实验设计手册,设计合成2条针对TBK1相关激酶活性缺失突变体和泛素样结构域突变体的引物,以实验室之前构建的TBK1野生型真核表达载体为模板,构建TBK1激酶活性缺失突变体和泛素样结构域突变体真核表达载体,分别命名为pcDNA3-Flag-TBK1(KD)、pcDNA3-F1ag-TBK1(△ULD).以LipofactAMINE2000转染试剂转染至293细胞中进行瞬时表达,利用萤光素酶实验检测2种TBK1突变体诱导β干扰素(IFN-β)转录的情况.结果:测序结果表明,TBK1相关激酶活性缺失突变体和泛素样结构域缺失突变体真核表达载体构建成功,Western印迹检测表明其在293细胞中获得有效表达；用萤光素酶报告基因实验检测,与野生型TBK1相比,其相关激酶活性缺失突变体和泛素样结构域缺失突变体诱导IFN-β转录激活的作用明显降低.结论:真核表达的TBK1相关激酶活性缺失突变体和泛素样结构域突变体具有相应的生物学活性,为研究其功能奠定了基础.