单纯疱疹病毒Ⅱ型多功能蛋白ICP27对Vero细胞凋亡作用的研究

发布时间：2018-05-11 10:41

本文选题：单纯疱疹病毒2型 + 多功能蛋白ICP27　；参考：《华南理工大学》2011年硕士论文

【摘要】：单纯疱疹病毒2型(HSV-2)是一种常见的人类至病菌,感染人腰部以下的皮肤和粘膜而主要引起生殖器疱疹。HSV-2感染人体后,便能在骶尾神经节内建立终身性的潜伏感染,而潜伏的病毒可因多种因素而激活,经外周感觉神经顺向移至皮肤粘膜表面发生皮损。目前,临床上用抗病毒药物来治疗HSV-2的感染,但不能彻底清除潜伏感染的病毒,且尚未研制出能安全有效预防HSV-2感染的疫苗。究其原因在于潜伏-复发的机制不明确。 ICP27是HSV IE基因编码的一种磷酸化蛋白,由512个氨基酸残基组成,分子量约为63kDa,是一种非常重要的病毒基因表达调节因子。在HSV感染期间表现出多种功能：在细胞质与细胞核间来回穿梭而将病毒mRNAs运输至细胞质中、抑制Ⅰ型干扰素信号而有利于病毒逃避宿主免疫监视、调节病毒一系列早期及晚期基因的表达而有利于病毒的增殖等。在潜伏感染期间,HSV基因为环状,而潜伏的病毒可因多种刺激而激活。为维持潜伏感染的状态,病毒感染细胞的凋亡作用应被阻止,LATs据报道能保护神经细胞而不发生凋亡。而ICP27在病毒的激活过程中可以激活p38与JNK信号通路而使细胞发生凋亡。因此,ICP27被认为在病毒的激活过程中起着重要的作用。为此,我们提取了病毒的基因组并以此为模板,用特异性的引物PCR扩增出ICP27片段。双酶切后,T4连接酶连接ICP27片段与真核表达质粒pEGFPC2而获得重组子,双酶切及测序鉴定后将重组质粒命为pEGFP-ICP27。接下来,将重组质粒pEGFP-ICP27瞬时转染Vero细胞,48h后于荧光显微镜下观察绿色荧光蛋白表达情况,并收集细胞提取总RNA及总蛋白,RT-PCR及western blot分别检测ICP27在Vero细胞中的转录及表达情况。PEGFP-ICP27在转染后的24h-48h之间,在Vero细胞中的表达量最大。 pEGFP-ICP27转染Vero细胞后,MTT法检测发现其活性明显低于转染pEGFPC2及空白转染的Vero细胞活性；Giemsa染色后发现转染pEGFP-ICP27的Vero细胞变圆,细胞核破裂,与之相对应的是凋亡执行蛋白Caspase-3的活性明显高于转染pEGFPC2及空白对照；荧光定量PCR检测Bax、Bcl-2 mRNAs的变化,转染重组质粒的Vero细胞中Bax mRNAs表达量略有上调,Bcl-2 mRNAs表达量降低,Bax/Bcl-2明显高于正常培养的Vero细胞及转染pEGFPC2质粒的Vero细胞。基于以上这些实验结果,我们可以认为HSV-2 ICP27具有促进细胞凋亡的作用,而这种作用可能是使Bax及Bcl-2在细胞中的比例发生变化而导致的。
[Abstract]:Herpes simplex virus type 2 (HSV-2) is a common human pathogen that infects skin and mucous membranes below the waist and causes mainly genital herpes. HSV-2 infection in the sacrococcygeal ganglion can establish a lifelong latent infection. The latent virus can be activated by many factors, and the skin lesions occur through peripheral sensory nerves. At present, antiviral drugs are used to treat HSV-2 infection in clinic, but the latent infection virus can not be completely eliminated, and no vaccine has been developed to prevent HSV-2 infection safely and effectively. The reason is that the mechanism of latent-recurrence is not clear. ICP27 is a phosphorylated protein encoded by HSV IE gene. It is composed of 512 amino acid residues and its molecular weight is about 63kDa. It is a very important regulatory factor for viral gene expression. During HSV infection, virus mRNAs was transported to the cytoplasm by shuttling back and forth between cytoplasm and nucleus, which inhibited the signal of interferon type I and facilitated the virus to escape from host immune surveillance. Regulating the expression of a series of early and late genes is beneficial to virus proliferation. During latent infection, HSV bases are circular and latent viruses can be activated by multiple stimuli. In order to maintain the latent infection, the apoptosis of virus-infected cells should be prevented by LATs, which is reported to protect nerve cells from apoptosis. ICP27 can activate p38 and JNK signaling pathway and induce apoptosis. Therefore, ICP 27 is thought to play an important role in the activation of viruses. Therefore, we extracted the genome of the virus and used it as a template to amplify the ICP27 fragment with specific primer PCR. The recombinant plasmid was obtained by ligating the ICP27 fragment with the eukaryotic expression plasmid pEGFPC2. The recombinant plasmid was designated as pEGFP-ICP27 after double enzyme digestion and sequencing. After transient transfection of recombinant plasmid pEGFP-ICP27 into Vero cells for 48 h, the expression of green fluorescent protein (GFP) was observed under fluorescence microscope. Total RNA, total protein RT-PCR and western blot were collected to detect the transcription and expression of ICP27 in Vero cells. PEGFP-ICP27 was expressed most in Vero cells after transfection with 24h-48h. The activity of pEGFP-ICP27 transfected Vero cells was significantly lower than that of Vero cells transfected with pEGFPC2 and blank transfected Vero cells. The Vero cells transfected with pEGFP-ICP27 became round and the nucleus ruptured. The activity of apoptotic executive protein Caspase-3 was significantly higher than that of transfected pEGFPC2 and blank control, and the changes of Bcl-2 mRNAs were detected by fluorescence quantitative PCR. The expression of Bax mRNAs in Vero cells transfected with recombinant plasmid was slightly up-regulated. The expression of Bax-Bax / Bcl-2 was significantly higher than that of Vero cells cultured in normal culture and Vero cells transfected with pEGFPC2 plasmids. Based on these results, we can conclude that HSV-2 ICP27 can promote apoptosis, which may be due to the change of the proportion of Bax and Bcl-2 in cells.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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