转录因子GATA-1与人巨细胞病毒UL111A基因5＇上游序列结合的实验研究  

Binding of transcription factor GATA-1 to 5' upstream sequence of human cytomegalovirus UL111 Agene

作　　者：田可港[1,2,4] 浮苗[1] 高艳[1] 彭颖[2] 李祥[2] 郑晓群[1] 徐志伟[1] 宋建华 吕建新[2] 

机构地区：[1]温州医学院附属第二医院实验诊断中心,浙江温州325027 [2]温州医学院浙江省医学遗传学重点实验室,浙江温州325035 [3]俄克拉荷马医学研究中心,美国俄克拉荷马73104 [4]宁波市鄞州人民医院

出　　处：《中国病理生理杂志》2012年第12期2244-2249,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81071365);温州市对外合作交流科技计划资助项目(No.H20090077)

摘　　要：目的:利用人巨细胞病毒(human cytomegalovirus,HCMV)潜伏及再激活感染THP-1细胞模型,研究细胞核内转录因子GATA-1与HCMV UL111A基因5'上游DNA序列的相互作用。方法:采用免疫印迹(Western blotting)技术定量分析HCMV潜伏及再激活感染时细胞核内转录因子GATA-1的表达;通过MATCH工具软件分析UL111A基因5'上游DNA序列,发现存在着11个GATA-1的结合位点,针对这11个GATA-1结合位点DNA序列设计9对引物,采用染色质免疫沉淀(chromatin immunoprecipitation,ChIP)及实时荧光定量PCR技术分析转录因子GATA-1与UL111A基因5'上游DNA序列的结合情况。结果:HCMV潜伏感染时UL111A基因未表达cmvIL-10,激活感染时表达cmvIL-10,潜伏感染时转录因子GATA-1的相对表达量高于激活感染;以GATA-1抗体特异性结合的DNA片段为模板,9对引物中共有5对扩增出目的条带;HCMV潜伏感染组和激活感染组5个位点转录因子GATA-1结合率分别为:-4.00±0.26、-4.50±0.33、-4.41±0.21、-3.61±0.20、-3.47±0.11和-1.13±0.07、-0.63±0.05、-1.10±0.07、-0.24±0.03和-0.14±0.01,差异均有统计学意义(P<0.05)。结论:HCMV感染时转录因子GATA-1可能通过与UL111A基因5’上游序列1119(-3760)位点、107(-4772)位点、609(-4270)位点、849(-4030)位点和2047(-2832)位点结合,调节UL111A基因的转录,参与HCMV潜伏与再激活感染的过程。AIM： To investigate the interactions between transcription factor GATA - 1 and 5＇ upstream DNA sequence of human cytomegalovirus（HCMV） UL111A gene using established HCMV latent and reactivated infection mod- els. METHODS ： The expression of transcription factor GATA - 1 in the process of HCMV latent and reactivated infections was determined by Western blotting. Sequence analysis revealed the presence of 11 consensus GATA - 1 binding sites on the 5 ＇upstream DNA sequence of UL111A gene using MATCH tool software. Moreover, 9 pairs of primers were designed for the core DNA sequence of the 11 binding sites. The bindings of GATA - 1 with 5＇ upstream DNA sequence of UL111A gene in HCMV latent and reactivated infections were assessed by real - time fluorescence quantitative PCR and chromatin immu- noprecipitation （CHIP）. RESULTS： UL111A gene did not express cmvIL -10 in latent infection but did in reactivated in- fection. The relative expression of transcription factor GATA - 1 was more in HCMV latent infection than that in reactivated infection. Five pairs of primers were used to amplify the target band when using the GATA - 1 antibody - specific binding DNA fragments as templates. The binding rates of GATA - 1 of 5 primers in HCMV latent infection group were - 4.00±0. 26, - 4.50±0.33, - 4.41±0.21, - 3.61±0.20 and - 3.47±0.11, while that in reactivated infection group were -1.13±0.07, -0.63±0. 05, - 1.10±0.07, -0. 24±0.03 and -0. 14±0. 01 （P 〈0. 05）. CONCLUSION： Transcription factor GATA - 1 mediates the UL111A transcription which is involved in HCMV latent and reactivated infec- tions by binding the HCMV UL111A gene 5＇ upstream sequence 107 （-4772） site, 609 （-4270） site, 849 （-4030） site, 1119 （-3760） site and 2047 （-2832） site during reactivated infection.

关 键 词：转录因子GATA-1 HCMV潜伏感染 UL111A 

分 类 号：R373.9[医药卫生—病原生物学]