摘要
目的构建人TRAF1真核表达载体,应用荧光素酶报告基因实验研究TRAF1对NF-κB报告基因活性的影响。方法采用PCR技术扩增出TRAF1编码基因,将其构到真核表达载体pFlag-CMV-2上。用脂质体将构建的真核表达载体瞬时转染HEK293细胞,用Western blot检测TRAF1蛋白是否表达。用荧光素酶报告基因实验验证其对NF-κB转录活性的影响。结果成功克隆TRAF1编码基因至相应表达载体上。构建的TRAF1真核表达载体使HEK293细胞高表达TRAF1蛋白;在肿瘤坏死因子(TNF-α)的刺激下可以抑制NF-κB的转录活性。结论TRAF1负调控NF-κB的活性,为进一步研究TRAF1的功能提供了线索。
Objective To construct the eukaryotic vector expressing TRAF1 cDNA and searched TRAF1 to active or inhibit NF-κB. Methods The coding gene of TRAF1, amplified by polymerase chain reaction, was cloned into pFLAG-CMV-2 vector. The vector was then transfected transiently into HEK,293 cell by lipofectamine. The expres- sion of TRAF1 was detected by Western blot . Then we used luciferase assay search TRAF1 how to influence NF- κB. Results The coding gene of TRAF1 was successfully cloned into pFLAG-CMV-2 vector, Abundant TRAF1 protein expressed in HEK293 cell transfected with pFLAG-CMV-2 vector. Luciferase assay elucidated TRAF1 may in- hibited NF-κB with TNF-a stimulation. Conclusion TRAF1 negatively regulates NF-κB activation,which provide a clue for further study on the functions of TRAF1.
出处
《安徽医科大学学报》
CAS
北大核心
2009年第2期147-150,共4页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学创新群体基金资助项目(编号:30621063)
国家高新技术研究发展计划(863计划)资助项目(编号:2006AA02A310)
国家重点基础研究发展规划(973计划)资助项目(编号:2006CB910802)
安徽省人才开发基金资助项目(编号:2002Z035)
关键词
NF-ΚB
基因
转录
肿瘤坏死因子-alpha
NF-kappaB
genes
transcription
tumor necrosis factor-alpha TRAF1 gene
eukaryotic expression vector
