【摘要】：新型環(huán)形病毒AGV2最早于2011年被檢測報道。AGV2基因組結構與雞傳染性貧血病毒CAV相似,同樣編碼VP1、VP2和VP3蛋白。與CAV的VP3蛋白類似,AGV2VP3蛋白也已被證明具有誘導腫瘤細胞凋亡的功能。CAV的VP3蛋白中108位的蘇氨酸等關鍵磷酸化位點已被證明,不僅與VP3在腫瘤細胞的核定位有關,而且與VP3誘導腫瘤細胞功能密切相關。那么在AGV2的VP3蛋白上是否也存在相應的潛在磷酸化位點?這些位點是否對AGV2 VP3蛋白的凋亡功能具有重要影響?為探究AGV2的VP3蛋白中可能的潛在磷酸化位點對AGV2 VP3凋亡功能影響,本研究在預測AGV2 VP3蛋白中4個潛在磷酸化位點的基礎上,進行了真核表達載體的構建,并評價了這4個潛在磷酸化位點對AGV2 VP3凋亡功能影響。一、AGV2VP3蛋白不同潛在磷酸化位點變體構建為了研究磷酸化位點對AGV2 VP3蛋白的凋亡功能作用,根據(jù)CAV的VP3蛋白中已驗證的與凋亡功能相關的磷酸化位點,利用overlap PCR技術將AGV2 VP3基因上的4個潛在磷酸化位點包括61位的蘇氨酸、90位的絲氨酸、91位的絲氨酸以及111位的蘇氨酸突變?yōu)楸彼?并克隆獲得了與EGFP蛋白融合的4個AGV2 VP3變體pCEGFP-AGV2 VP3T61A、pCEGFP-AGV2VP3S90A、pCEGFP-AGV2VP3S91A 和 pCEGFP-AGV2 VP3T111A。通過轉染293T細胞,分別用抗AGV2VP3抗體以及GFP抗體對4個VP3變體的表達進行了 Western Blot分析。結果發(fā)現(xiàn),四個變體均能很好的表達VP3蛋白。在Western Blot中可見42kD處有特異性條帶。四個潛在磷酸化位點VP3變體的構建及表達為進一步研究磷酸化位點對AGV2 VP3凋亡功能的影響打下了基礎。二、不同潛在磷酸化位點對AGV2VP3凋亡功能影響為檢測潛在磷酸化位點對AGV2 VP3凋亡功能的影響,將獲得的表達4個潛在磷酸化位點變體的 VP3 的陽性質粒 pCEGFP-AGV2 VP3T61A、pCEGFP-AGV2 VP3S90A、pCEGFP-AGV2 VP3S91A 和 pCEGFP-AGV2 VP3T111A,以及野生型 pCEGFP-AGV2 VP3分別轉染人結腸癌細胞HCT116、正常雞胚成纖維細胞CEF以及DF1細胞。通過激光共聚焦顯微鏡觀察發(fā)現(xiàn),AGV2VP3及其變體在HCT116以及DF1細胞均定位于細胞核內,且呈散在的點狀分布,類似凋亡小體;而在CEF中在胞漿及胞核均有表達。利用Annexin V-PE/7-AAD 流式細胞術檢測 VP3 凋亡發(fā)現(xiàn),pCEGFP-AGV2VP3T61A、pCEGFP-AGV2 VP3S90A、pCEGFP-AGV2 VP3S91A 和 pCEGFP-AGV2 VP3T111A 對 HCT116 細胞誘導凋亡比率比對照EGFP質粒高10%-20%。在檢測caspase活性時發(fā)現(xiàn),AGV2 VP3及其各變體的活性值是對照EGFP的1.5倍到2倍。這些結果表明,所檢測的4個潛在磷酸化位點對AGV2 VP3的細胞內定位及其凋亡功能的影響不大。提示AGV2 VP3蛋白中其它磷酸化位點或不同磷酸化位點的協(xié)同作用對AGV2 VP3的凋亡功能影響有待進一步探究。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65

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