文章摘要
钱宝梅,王文博,罗 灿,张 婧,赵 聃,王迎伟,邱 文.大鼠MIP⁃1α基因启动子荧光素酶报告质粒的构建及其IRF⁃8结合元件的初步鉴定[J].南京医科大学学报,2019,(1):10~15
大鼠MIP⁃1α基因启动子荧光素酶报告质粒的构建及其IRF⁃8结合元件的初步鉴定
Construction of luciferase reporter plasmids of rat MIP⁃1α promoter and initial identification of IRF⁃8 binding element
投稿时间:2018-10-24  
DOI:10.7655/NYDXBNS20190102
中文关键词: 巨噬细胞炎性蛋白⁃1α(MIP⁃1α)  干扰素调节因子⁃8(IRF⁃8)  启动子
英文关键词: macrophage inflammatory protein⁃1α(MIP1⁃α)  interferon regulatory factor⁃8(IRF⁃8)  promoter
基金项目:国家自然科学基金(31470853,81471626);南京医科大学优秀中青年教师支持计划
作者单位
钱宝梅 南京医科大学基础医学院免疫学系江苏 南京 211166 
王文博 南京医科大学基础医学院免疫学系江苏 南京 211166 
罗 灿 南京医科大学基础医学院免疫学系江苏 南京 211166 
张 婧 南京医科大学基础医学院免疫学系江苏 南京 211166 
赵 聃 南京医科大学基础医学院免疫学系江苏 南京 211166 
王迎伟 南京医科大学基础医学院免疫学系江苏 南京 211166 
邱 文 南京医科大学基础医学院免疫学系江苏 南京 211166 
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中文摘要:
      目的:构建大鼠巨噬细胞炎性蛋白?1α(macrophage inflammatory protein?1α,MIP?1α)基因启动子(全长和截短)荧光素酶报告质粒,并观察在人胚肾细胞(HEK?293T)中过表达干扰素调节因子?8(interferon regulatory factor?8,IRF?8)对MIP?1α基因启动活性的影响,同时筛选其可能的IRF?8结合元件。方法:采用PCR技术,扩增出大鼠MIP?1α基因启动子序列,将MIP?1α基因启动子插入到荧光素酶报告基因载体pGL3?basic中,构建MIP?1α基因启动子全长荧光素酶报告质粒(pGL3?MIP?1α?FL)。将上述pGL3?MIP?1α?FL和本课题组已构建的大鼠IRF?8过表达质粒(pIRES2?IRF?8)共转染HEK?293T细胞,检测细胞内荧光素酶活性,确定IRF?8对MIP?1α基因的启动作用。同时,应用生物信息学软件预测MIP?1α基因启动子上IRF?8的结合元件,并据此构建3个MIP?1α基因启动子截短的荧光素酶报告质粒(pGL3?MIP?1α?1~3)。将上述MIP?1α基因启动子全长和各截短的荧光素酶报告质粒和IRF?8过表达质粒共转染HEK?293T细胞,再测定荧光素酶活性,初步确定IRF?8的结合元件。结果:菌液PCR及核酸测序证实,上述pGL3?MIP?1α?FL(-1 400~+94 nt)质粒构建成功。将pGL3?MIP?1α?FL和pIRES2?IRF?8共转染HEK?293T后发现,过表达IRF?8可显著增加MIP?1α基因启动子活性。应用生物信息学软件预测发现MIP?1α基因启动子上IRF?8的结合元件(-1 157~-1 144nt、-740~-734 nt、-683~-670 nt、-365~-359 nt、-249~-236 nt),并据此构建3个MIP?1α基因启动子截短的荧光素酶报告质粒,即pGL3?MIP?1α?1(-453~+94 nt)、pGL3?MIP?1α?2(-352~+94 nt)和pGL3?MIP?1α?3(-3~+94 nt)。将pGL3?MIP?1α?FL、pGL3?MIP?1α?1~3和pIRES2?IRF?8共转染HEK?293T后发现,pGL3?MIP?1α?3的启动活性显著低于pGL3?MIP?1α?FL、pGL3?MIP?1α?1和pGL3?MIP?1α?2。提示IRF?8可能结合在大鼠MIP?1α基因启动子的-352~-3 nt区域的IRF?8结合元件(-249~-236 nt)上。结论:本实验成功构建了大鼠MIP?1α基因启动子全长及截短荧光素酶报告质粒,并初步筛查出IRF?8在MIP?1α基因启动子上的可能结合元件,为后续研究奠定了基础。
英文摘要:
      Objective:To construct luciferase reporter plasmids of full?length and truncated promotors of rat macrophage inflammatory protein?1α(MIP?1α)gene and detect their activity in HEK?293T cells in response to interferon regulatory factor?8(IRF?8)overexpression,screening the possible binding elements for IRF?8. Methods:Rat MIP?1α promoter was amplified by PCR and cloned into the luciferase reporter plasmid(pGL3?basic). The recombinant plasmid(pGL3?MIP?1α?FL)and rat IRF?8 overexpression plasmid(pIRES2?IRF?8)were co?transfected into HEK?293T cells and then the luciferase activity was detected to determine the role of IRF?8 in MIP?1α gene transcription. Meanwhile,the potential IRF?8 binding elements within MIP?1α promoter were predicted by using bioinformatics software. Based on the predicted results,three luciferase reporter plasmids of truncated MIP?1α gene promotor(pGL3?MIP?1α?1~3)were constructed. The promoter luciferase reporter plasmids of pGL3?MIP?1α?FL or pGL3?MIP?1α?1~3 and the plasmid of pIRES2?IRF?8 were co?transfected into HEK?293T cells. Then,the luciferase activity was detected to screen the IRF?8 binding elements. Results:It was verified that pGL3?MIP?1α?FL(-1 400~+94 nt)plasmid was constructed correctly by PCR analysis and nucleotide sequencing. The plasmids of pGL3?MIP?1α?FL and pIRES2?IRF?8 were co?transfected into HEK?293T cells,and then the luciferase activity of MIP?1α gene promotor was markedly increased in response to IRF?8 overexpression. The potential IRF?8 binding elements(-1 157~ -1 144 nt,-740~ -734 nt,-683~ -670 nt,-365~-359 nt,and -249 ~ -236 nt)within MIP?1α promoter were predicted by using bioinformatics software. Based on the predicted results,we constructed three luciferase reporter plasmids of truncated MIP?1α gene promotor,namely pGL3?MIP?1α?1(-453 ~ +94 nt),pGL3?MIP?1α?2(-352 ~ +94 nt)and pGL3?MIP?1α?3(-3 ~ +94 nt). The plasmids of pGL3?MIP?1α?FL or pGL3?MIP?1α?1~3 and pIRES2?IRF?8 were co?transfected into HEK?293T cells,and the result displayed that the activity of pGL3?MIP?1α?3 was much lower than that in pGL3?MIP?1α?FL,pGL3?MIP?1α?1 and pGL3?MIP?1α?2,indicating that the region of rat MIP?1α promoter(-352 ~ -3 nt)might contain an IRF?8 binding element(-249 ~ -236 nt). Conclusion:The rat full?length and truncated rat MIP?1α promotor luciferase reporter plasmids were constructed successfully,and the possible IRF?8 binding element was found,which could be beneficial to further studies.
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