【作者】 陈琳；

【导师】 万艳平；

【作者基本信息】 南华大学 ， 病原生物学， 2008， 硕士

【摘要】 目的:构建人巨细胞病毒(HCMV)IE1蛋白与增强型绿色荧光蛋白EGFP融合蛋白真核细胞表达载体pEGFP-C1/IE1,将其转染入THP-1巨噬细胞,初步探讨GFP-HCMV IE1融合蛋白瞬时高表达对THP-1巨噬细胞分泌细胞因子活性及其凋亡的影响,为进一步研究IE1蛋白在HCMV致病机制中的作用奠定实验基础。方法:(1)用HindⅢ和BamHⅠ双酶切pNEB193/IE1质粒,将IE1基因片段亚克隆入pcDNA3.1(+)载体,双酶切鉴定及测序分析; SalⅠ和BamHⅠ双酶切质粒pcDNA3.1(+)/IE1,然后将目的基因亚克隆入pEGFP-C1,构建真核表达载体pEGFP-C1/IE1。(2)将真核表达载体pEGFP-C1/IE1与pEGFP-C1分别转染THP-1巨噬细胞,实验分3组:巨噬细胞空白对照组(Control组);pEGFP-C1转染巨噬细胞组(GFP组);pEGFP-C1/IE1转染巨噬细胞组(GFP-IE1组)。转染48h后,通过Western-blotting和倒置荧光显微镜检测GFP-HCMVIE1融合蛋白或GFP的表达与定位。(3)利用ELISA分别检测12h、24h、48h各组细胞培养上清中TNF-α和IL-1β的含量;采用RT-PCR检测其mRNA的表达;收集巨噬细胞经PI染色后流式细胞术(FCM)检测其凋亡率。(4)利用统计软件SPSS13.0分析实验数据。结果:(1) IE1基因亚克隆至pcDNA3.1(+)载体上后,经双酶切及测序分析,所克隆的目的片段与GenBank上公布的HCMV IE1基因序列完全一致,然后将目的片段亚克隆入pEGFP-C1中,双酶切结果显示构建了真核表达载体pEGFP-C1/IE1,且GFP-IE1融合蛋白在THP-1巨噬细胞内瞬时表达并定位于细胞核,GFP在整个细胞中均匀表达。Western-blotting结果表明在THP-1巨噬细胞中表达了分子量约101.4kD的融合蛋白。(2)转染12h时,GFP-IE1组培养上清中TNF-α、IL-1β含量明显升高,随着时间的递增,2种细胞因子分泌量呈上升趋势,与空白对照组或GFP组比较差异有显著性(P<0.01)。GFP瞬时表达对2种细胞因子的分泌影响差异无显著性(P>0.05)。GFP-IE1组TNF-α和IL-1βmRNA表达水平均增加。(3)转染后48h,GFP-IE1组巨噬细胞凋亡率明显升高,与空白对照组或GFP组比较差异有显著性(P<0.01),而GFP组巨噬细胞凋亡率与对照组比较差异无显著性(P>0.05)。结论:(1)成功构建了真核表达载体pEGFP-C1/IE1 ,且能在THP-1巨噬细胞中表达GFP-HCMV IE1融合蛋白并定位于细胞核。(2) GFP-HCMV IE1融合蛋白瞬时高表达上调THP-1巨噬细胞分泌TNF-α和IL-1β。(3) GFP-HCMV IE1融合蛋白瞬时高表达诱导THP-1巨噬细胞凋亡。更多还原

【Abstract】 Objective: To study the effect of over-expression of GFP-IE1 fusion protein on secretion and apoptosis of THP-1-macrophage , and to provide the experimental basis for the further studies on the pathogenesis of IE1 protein from Human cytomegalovirus （HCMV）.Methods:（1） HCMV IE1 gene was excised from pNEB193-IE1 with BamHⅠ/HindⅢand subcloned into pCDNA3.1（+）. The reconstructed plasmid was confirmed by restriction enzyme and DNA sequencing respectively. Then the pCDNA3.1（+）/IE1 was digested with SalⅠ/BamHⅠand IE1 gene product was subcloned into pEGFP-C1 to reconstruct the pEGFP-C1/IE1.（2） The plasmid pEGFP-C1/IE1 or pEGFP-C1 was transfected into THP-1-macrophage respectively. The experiment was divided into three groups, namely, control group, GFP group, GFP-IE1 group. Forty-eight hours after transfection, the expression and localization of GFP or GFP-IE1 was observed under the fluorescent microscope.（3） The level of TNF-αor IL-1βin the cells media was examined by ELISA at 12h, 24h, 48h, respectively. The mRNA expression of them was analyzed by RT-PCR. Cells undergoing apoptosis were determined by flow cytometry using the propidium iodide staining method （PI）.（4） All of data were analyzed by SPSS13.0.Results:（1） The size and sequence of IE1 gene in pCDNA3.1（+）/IE1 was identical with that in GenBank （Pubmed NC₀01347）, using restriction enzyme analysis and sequencing. Then the eukaryotic expression vector pEGFP-C1/IE1 was successfully constructed as described above. At 48 h after transfection of THP-1-macrophage with pEGFP-C1/IE1 or pEGFP-C1, GFP could be detected in whole cells using fluorescent microscope, Whereas GFP-IE1 fusion protein only located in nuclei. Western blot result showed that the bands with molecular weight of about 101.4KD were consistent with GFP-IE1. （2） Twelve hours after transfection, the level of TNF-αor IL-1βof GFP-IE1 group obviously increased. As time increases, two kinds of cytokines secretion is the rising trend. GFP-IE1 group had an obvious difference compared with the control group or GFP group（P<0.01）, but no significant difference existed between GFP group and the control group（P>0.05）. TNF-αor IL-1βmRNA expression enhanced in macrophages transfected with pEGFP-C1/IE1.（3） At 48h after transfection, flow cytometry assays demonstrated that GFP had not remarkable difference compared with control group （P>0.05）. While there was remarkable difference on the apoptosis rates of macrophages between GFP-IE1 and control group or GFP group （P <0.01）.Conclusion:（1） The recombinant eukaryotic expression vector pEGFP-C1/IE1 was successfully constructed, and GFP-IE1 fusion protein could express correctly in THP-1-macrophage. GFP-IE1 fusion protein only located in nuclei.（2） Transient expression of GFP-IE1 fusion protein up-regulated TNF-αand IL-1βsecretion of THP-1-macrophage.（3） Transient expression of GFP-IE1 fusion protein induced the apoptosis of THP-1 -macrophage.更多还原