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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。
OBJECTIVE: To investigate the interaction between nuclear transcription factor GATA-1 and the 5 ’upstream DNA sequence of HCMV UL111A gene in THP-1 cells by latent and reactivation of human cytomegalovirus (HCMV). Methods: Western blotting was used to quantitatively analyze the expression of GATA-1 in latent nucleus and reactivation of HCMV. The DNA sequence of 5 ’upstream of UL111A gene was analyzed by MATCH software and 11 GATA-1 Nine pairs of primers were designed according to the DNA sequence of the 11 GATA-1 binding sites. The transcription factor GATA-1 and UL111A gene 5 ’were analyzed by chromatin immunoprecipitation (ChIP) and real-time fluorescence quantitative PCR Upstream DNA sequence binding. Results: Under the latent infection of HCMV, the UL111A gene did not express cmvIL-10, and the expression of cmvIL-10 was found in the activated infection. The relative expression level of GATA-1 in latent infection was higher than that in activated infection. The DNA fragment with specific binding of GATA- As a template, a total of 5 pairs of 9 pairs of primers amplified the target bands. The binding rates of GATA-1 to 5 sites in HCMV latent infection group and activated infection group were -4.00 ± 0.26 and -4.50 ± 0.33, respectively. 4.41 ± 0.21, -3.61 ± 0.20, -3.47 ± 0.11 and -1.13 ± 0.07, -0.63 ± 0.05, -1.10 ± 0.07, -0.24 ± 0.03 and -0.14 ± 0.01, the difference was statistically significant (P <0.05) . CONCLUSIONS: GATA-1 may be associated with the site of 1119 (-3760), 107 (-4772), 609 (-4270), 849 (-4030) in HCMV infection And 2047 (-2832) sites, regulating UL111A gene transcription, involved in HCMV latent and reactivation of the infection process.