【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p GL3-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)、p GL3-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,p GL3-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。更多还原

【Abstract】 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-293 T 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 clonedinto 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-293 T 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 usingbioinformatics 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 p GL3-MIP-1α-1～3 and the plasmidof pIRES2-IRF-8 were co-transfected into HEK-293 T 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 andnucleotide sequencing. The plasmids of pGL3-MIP-1α-FL and pIRES2-IRF-8 were co-transfected into HEK-293 T cells,and then theluciferase activity of MIP-1α gene promotor was markedly increased in response to IRF-8 overexpression. The potential IRF-8 bindingelements(-1 157～-1 144 nt,-740～-734 nt,-683～-670 nt,-365～-359 nt,and-249 ～-236 nt)within MIP-1α promoter werepredicted by using bioinformatics software. Based on the predicted results,we constructed three luciferase reporter plasmids oftruncated 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 p IRES2-IRF-8 were co-transfected into HEK-293 T cells,andthe 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 thepossible IRF-8 binding element was found,which could be beneficial to further studies.更多还原