潜在磷酸化位点对AGV2 VP3凋亡功能的影响
发布时间：2018-07-16 22:54
【摘要】：新型环形病毒AGV2最早于2011年被检测报道。AGV2基因组结构与鸡传染性贫血病毒CAV相似,同样编码VP1、VP2和VP3蛋白。与CAV的VP3蛋白类似,AGV2VP3蛋白也已被证明具有诱导肿瘤细胞凋亡的功能。CAV的VP3蛋白中108位的苏氨酸等关键磷酸化位点已被证明,不仅与VP3在肿瘤细胞的核定位有关,而且与VP3诱导肿瘤细胞功能密切相关。那么在AGV2的VP3蛋白上是否也存在相应的潜在磷酸化位点?这些位点是否对AGV2 VP3蛋白的凋亡功能具有重要影响?为探究AGV2的VP3蛋白中可能的潜在磷酸化位点对AGV2 VP3凋亡功能影响,本研究在预测AGV2 VP3蛋白中4个潜在磷酸化位点的基础上,进行了真核表达载体的构建,并评价了这4个潜在磷酸化位点对AGV2 VP3凋亡功能影响。一、AGV2VP3蛋白不同潜在磷酸化位点变体构建为了研究磷酸化位点对AGV2 VP3蛋白的凋亡功能作用,根据CAV的VP3蛋白中已验证的与凋亡功能相关的磷酸化位点,利用overlap PCR技术将AGV2 VP3基因上的4个潜在磷酸化位点包括61位的苏氨酸、90位的丝氨酸、91位的丝氨酸以及111位的苏氨酸突变为丙氨酸,并克隆获得了与EGFP蛋白融合的4个AGV2 VP3变体pCEGFP-AGV2 VP3T61A、pCEGFP-AGV2VP3S90A、pCEGFP-AGV2VP3S91A 和 pCEGFP-AGV2 VP3T111A。通过转染293T细胞,分别用抗AGV2VP3抗体以及GFP抗体对4个VP3变体的表达进行了 Western Blot分析。结果发现,四个变体均能很好的表达VP3蛋白。在Western Blot中可见42kD处有特异性条带。四个潜在磷酸化位点VP3变体的构建及表达为进一步研究磷酸化位点对AGV2 VP3凋亡功能的影响打下了基础。二、不同潜在磷酸化位点对AGV2VP3凋亡功能影响为检测潜在磷酸化位点对AGV2 VP3凋亡功能的影响,将获得的表达4个潜在磷酸化位点变体的 VP3 的阳性质粒 pCEGFP-AGV2 VP3T61A、pCEGFP-AGV2 VP3S90A、pCEGFP-AGV2 VP3S91A 和 pCEGFP-AGV2 VP3T111A,以及野生型 pCEGFP-AGV2 VP3分别转染人结肠癌细胞HCT116、正常鸡胚成纤维细胞CEF以及DF1细胞。通过激光共聚焦显微镜观察发现,AGV2VP3及其变体在HCT116以及DF1细胞均定位于细胞核内,且呈散在的点状分布,类似凋亡小体;而在CEF中在胞浆及胞核均有表达。利用Annexin V-PE/7-AAD 流式细胞术检测 VP3 凋亡发现,pCEGFP-AGV2VP3T61A、pCEGFP-AGV2 VP3S90A、pCEGFP-AGV2 VP3S91A 和 pCEGFP-AGV2 VP3T111A 对 HCT116 细胞诱导凋亡比率比对照EGFP质粒高10%-20%。在检测caspase活性时发现,AGV2 VP3及其各变体的活性值是对照EGFP的1.5倍到2倍。这些结果表明,所检测的4个潜在磷酸化位点对AGV2 VP3的细胞内定位及其凋亡功能的影响不大。提示AGV2 VP3蛋白中其它磷酸化位点或不同磷酸化位点的协同作用对AGV2 VP3的凋亡功能影响有待进一步探究。
[Abstract]:A novel annular virus AGV2 was first detected in 2011. The genome structure of AGV2 is similar to that of chicken infectious anemia virus (CAV), and it also encodes VP1 VP2 and VP3 proteins. Similar to the VP3 protein of CAV, AGV2VP3 protein has also been proved to have the function of inducing apoptosis of tumor cells. The key phosphorylation sites such as threonine in the VP3 protein of CAV have been proved to be not only related to the nuclear localization of VP3 in tumor cells. Moreover, it is closely related to the function of tumor cells induced by VP3. Is there a corresponding potential phosphorylation site on the VP3 protein of AGV2? Do these sites have an important effect on the apoptotic function of AGV2 VP3 protein? In order to investigate the effect of potential phosphorylation sites in AGV2 VP3 protein on the apoptotic function of AGV2 VP3, the eukaryotic expression vector was constructed based on the prediction of four potential phosphorylation sites in AGV2 VP3 protein. The effects of these four potential phosphorylation sites on the apoptotic function of AGV 2 VP3 were evaluated. A variant of different potential phosphorylation sites of AGV2VP3 protein was constructed to study the effect of phosphorylation sites on the apoptotic function of AGV2VP3 protein. The four potential phosphorylation sites of AGV2VP3 gene were mutated to alanine by overlap technique, including 61 sites of threonine, 90 position of serine, 91 position of serine, and 111 position of threonine, respectively. Four AGV2VP3 variants pCEGFP-AGV2VP3T61A, pCEGFP-AGV2VP3S90A, pCEGFP-AGV2VP3S91A and pCEGFP-AGV2VP3T111A were cloned. After transfection of 293T cells, the expression of four VP3 variants was analyzed by Western blot with anti-AGV2VP3 antibody and GFP antibody respectively. The results showed that the four variants could express VP3 protein well. A specific band of 42 KD was found in Western Blot. The construction and expression of four potential phosphorylation site VP3 variants lay a foundation for further study of the effects of phosphorylation sites on the apoptosis function of AGV2 VP3. Secondly, the effects of different potential phosphorylation sites on the apoptosis function of AGV2VP3 were determined by detecting the effects of potential phosphorylation sites on the apoptotic function of AGV2VP3. The positive plasmids pCEGFP-AGV2VP3T61A, pCEGFP-AGV2VP3S90A91A, pCEGFP-AGV2VP3S91A and pCEGFP-AGV2VP3T111A, wild type pCEGFP-AGV2V2VP3 were transfected into human colon cancer cell line HCT116, normal chicken embryo fibroblast CEF and DF1 cells, respectively. By laser confocal microscopy, it was found that AGV2VP3 and its variants were located in the nucleus of HCT116 and DF1 cells, and distributed in scattered spots, similar to apoptotic corpuscles, but expressed in cytoplasm and nucleus of CEF. Annexin V-PE-7-AAD flow cytometry was used to detect the apoptosis of HCT116 cells. It was found that pCEGFP-AGV2VP3T61A, pCEGFP-AGV2VP3S91A and pCEGFP-AGV2VP3T111A could induce apoptosis in HCT116 cells. It was found that the activity of AGV2VP3 and its variants was 1.5 to 2 times higher than that of the control. These results suggest that the four potential phosphorylation sites detected have little effect on the intracellular localization and apoptotic function of AGV2VP3. It is suggested that the synergistic effects of other phosphorylation sites or different phosphorylation sites in AGV2 VP3 protein on the apoptosis function of AGV2 VP3 protein need to be further explored.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：2127926
[Abstract]:A novel annular virus AGV2 was first detected in 2011. The genome structure of AGV2 is similar to that of chicken infectious anemia virus (CAV), and it also encodes VP1 VP2 and VP3 proteins. Similar to the VP3 protein of CAV, AGV2VP3 protein has also been proved to have the function of inducing apoptosis of tumor cells. The key phosphorylation sites such as threonine in the VP3 protein of CAV have been proved to be not only related to the nuclear localization of VP3 in tumor cells. Moreover, it is closely related to the function of tumor cells induced by VP3. Is there a corresponding potential phosphorylation site on the VP3 protein of AGV2? Do these sites have an important effect on the apoptotic function of AGV2 VP3 protein? In order to investigate the effect of potential phosphorylation sites in AGV2 VP3 protein on the apoptotic function of AGV2 VP3, the eukaryotic expression vector was constructed based on the prediction of four potential phosphorylation sites in AGV2 VP3 protein. The effects of these four potential phosphorylation sites on the apoptotic function of AGV 2 VP3 were evaluated. A variant of different potential phosphorylation sites of AGV2VP3 protein was constructed to study the effect of phosphorylation sites on the apoptotic function of AGV2VP3 protein. The four potential phosphorylation sites of AGV2VP3 gene were mutated to alanine by overlap technique, including 61 sites of threonine, 90 position of serine, 91 position of serine, and 111 position of threonine, respectively. Four AGV2VP3 variants pCEGFP-AGV2VP3T61A, pCEGFP-AGV2VP3S90A, pCEGFP-AGV2VP3S91A and pCEGFP-AGV2VP3T111A were cloned. After transfection of 293T cells, the expression of four VP3 variants was analyzed by Western blot with anti-AGV2VP3 antibody and GFP antibody respectively. The results showed that the four variants could express VP3 protein well. A specific band of 42 KD was found in Western Blot. The construction and expression of four potential phosphorylation site VP3 variants lay a foundation for further study of the effects of phosphorylation sites on the apoptosis function of AGV2 VP3. Secondly, the effects of different potential phosphorylation sites on the apoptosis function of AGV2VP3 were determined by detecting the effects of potential phosphorylation sites on the apoptotic function of AGV2VP3. The positive plasmids pCEGFP-AGV2VP3T61A, pCEGFP-AGV2VP3S90A91A, pCEGFP-AGV2VP3S91A and pCEGFP-AGV2VP3T111A, wild type pCEGFP-AGV2V2VP3 were transfected into human colon cancer cell line HCT116, normal chicken embryo fibroblast CEF and DF1 cells, respectively. By laser confocal microscopy, it was found that AGV2VP3 and its variants were located in the nucleus of HCT116 and DF1 cells, and distributed in scattered spots, similar to apoptotic corpuscles, but expressed in cytoplasm and nucleus of CEF. Annexin V-PE-7-AAD flow cytometry was used to detect the apoptosis of HCT116 cells. It was found that pCEGFP-AGV2VP3T61A, pCEGFP-AGV2VP3S91A and pCEGFP-AGV2VP3T111A could induce apoptosis in HCT116 cells. It was found that the activity of AGV2VP3 and its variants was 1.5 to 2 times higher than that of the control. These results suggest that the four potential phosphorylation sites detected have little effect on the intracellular localization and apoptotic function of AGV2VP3. It is suggested that the synergistic effects of other phosphorylation sites or different phosphorylation sites in AGV2 VP3 protein on the apoptosis function of AGV2 VP3 protein need to be further explored.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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