文章摘要
王文博,钱宝梅,罗 灿,彭明玉,张 婧,赵 聃,王迎伟,邱 文,季明德.NF⁃κB p65调控IRF⁃8基因启动子活性及其启动子结合元件的初步鉴定[J].南京医科大学学报,2020,(5):638~644
NF⁃κB p65调控IRF⁃8基因启动子活性及其启动子结合元件的初步鉴定
The regulatory effects of NF⁃kB p65 on IRF⁃8 gene promoter activity and initial identification of its binding elements
投稿时间:2019-11-17  
DOI:10.7655/NYDXBNS20200505
中文关键词: 核因子⁃κB p65(NF⁃κB p65)  干扰素调节因子⁃8(IRF⁃8)  启动子  结合元件
英文关键词: nuclear factor⁃κB p65(NF⁃κB p65)  interferon regulatory factor⁃8(IRF⁃8)  promoter  binding elements
基金项目:国家自然科学基金(31470853,81603358,31770934,81971468)
作者单位
王文博 南京医科大学免疫学系江苏 南京 211166 
钱宝梅 南京医科大学免疫学系江苏 南京 211166 
罗 灿 南京医科大学免疫学系江苏 南京 211166 
彭明玉 南京医科大学免疫学系江苏 南京 211166 
张 婧 南京医科大学免疫学系江苏 南京 211166 
赵 聃 南京医科大学免疫学系江苏 南京 211166 
王迎伟 南京医科大学免疫学系江苏 南京 211166 
邱 文 南京医科大学免疫学系江苏 南京 211166 
季明德 江苏省中医院检验科江苏 南京 210006 
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中文摘要:
      目的:探讨大鼠核因子?κB(nuclear factor?κB,NF?κB)p65亚基在细胞内过表达和其活性变化对干扰素调节因子?8(interferon regulatory factor?8,IRF?8)基因启动的影响,并初步筛查IRF?8启动子上可能的p65结合元件。方法:采用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增大鼠p65基因蛋白编码区(complete sequence coding,CDS)序列,将其插入到空载pIRES2?EGFP质粒中,构建大鼠野生型(wild type,WT)p65过表达质粒(pIRES2?p65 WT)。在此基础上,将p65第535位丝氨酸(serine,S)突变为天冬氨酸(aspartic,D)或丙氨酸(alanine,A),分别构建p65持续活化突变型质粒(pIRES2?p65 S535D)和p65显性负性突变型质粒(pIRES2?p65 S535A)。之后应用生物信息学软件预测IRF?8基因启动子区p65的结合元件,并据此构建IRF?8启动子全长(full?length,FL)和3个截短的荧光素酶报告质粒,即pGL3?IRF?8?FL(-1 892 ~ +174 nt)、pGL3?IRF?8?1(-1 360 ~ +174 nt)、pGL3?IRF?8?2(-752 ~ +174 nt)和pGL3?IRF?8?3(-68 ~ +174 nt)。将上述质粒行不同组合共转染人胚肾293T(human embryonic kidney 293T,HEK?293T)细胞,Western blot和荧光素酶实验分别检查p65的表达和IRF?8的启动子活性,并分析IRF?8启动子区可能的p65结合元件。结果:菌液PCR及测序证实上述质粒构建成功。分别将pIRES2?p65 WT、pIRES2?p65 S535D、pIRES2?p65 S535A和pGL3?IRF?8?FL共转染HEK?293T,发现过表达pIRES2?p65 WT或pIRES2?p65 S535D均可明显增加IRF?8启动子活性,且以pIRES2?p65 S535D更为显著;而过表达pIRES2?p65 S535A后,IRF?8启动子活性无明显变化。将pGL3?IRF?8?FL、pGL3?IRF?8?1~3和pIRES2?p65 S535D共转染HEK?293T后发现,pGL3?IRF?8?3的启动子活性显著低于pGL3?IRF?8?FL、pGL3?IRF?8?1和pGL3?IRF?8?2,提示大鼠IRF?8启动子-752~68 nt区域可能存在p65结合元件。结论:在HEK?293T细胞内过表达野生型或持续活化突变型p65可显著促进IRF?8基因的启动,且p65与IRF?8启动子的结合元件可能位于-752~-68 nt部位。
英文摘要:
      Objective:This study aims to investigate the effect of rat nuclear factor?κB(NF?κB)p65 subunit over?expression and its activity changes on the gene promoter activity of interferon regulatory factor?8(IRF?8),and initially screen the possible p65?binding elements within IRF?8 promoter. Methods:To construct the rat wild type p65 over?expression plasmid(pIRES2?p65 WT),complete sequence coding(CDS)of rat p65 was amplified by polymerase chain reaction(PCR) and cloned into pIRES2?EGFP. Then,S535D and S535A mutation was done respectively based on the wild type p65 over?expression plasmid to construct p65 constitutively active mutant(pIRES2?p65 S535D)and p65 dominant negative mutant(pIRES2?p65 S535A). The potential p65?binding elements within IRF?8 promoter were predicted by using bioinformatics software. Based on the predicted results,luciferase reporter plasmids of full?length(FL)and three truncated IRF?8 gene promoter were constructed,namely pGL3?IRF?8?FL(-1 892~+174 nt),pGL3?IRF?8?1(-1 360~+174 nt),pGL3?IRF?8?2(-752~+174 nt),pGL3?IRF?8?3(-68~+174 nt). The above?mentioned plasmids were co?transfected into human embryonic kidney 293T(HEK?293T)cells in different groups. Then,the expression level of p65 was detected by Western blot,and the promoter activity of IRF?8 was detected by luciferase experiment to screen the p65?binding elements. Results:It was verified that above?mentioned plasmid was constructed correctly by PCR analysis and nucleotide sequencing. The plasmids of pIRES2?p65 WT,pIRES2?p65 S535D,pIRES2?p65 S535A were respectively transfected into HEK?293T cells together with pGL3?IRF?8?FL. The luciferase results showed that the activity of IRF?8 promoter was markedly increased in response to pIRES2?p65 WT and pIRES2?p65 S535D,especially the later. However,there was no significant change of IRF?8 promoter activity after over?expression of pIRES2?p65 S535A. The plasmids of pGL3?IRF?8?FL or pGL3?IRF?8?1~3 and pIRES2?p65 were co?transfected into HEK?293T cells,and the result displayed that the activity of pGL3?IRF?8?3 was much lower than that of pGL3?IRF?8?FL,pGL3?IRF?8?1 and pGL3?IRF?8?2,indicating that the region of rat IRF?8 promoter(-752~-68 nt) might contain p65?binding elements. Conclusion:Over?expression of wild?type or continuously activated mutant p65 in HEK?293T cells can significantly promote the activity of IRF?8 promoter,and the p65?binding elements in IRF?8 promoter might be located in the -752~-68 nt region.
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