鸭胚成纤维细胞翻译延伸因子对鹅细小病毒增殖的影响

发布时间：2019-04-29 11:32

【摘要】：鹅细小病毒(Goose parvovirus,GPV)病,又称小鹅瘟,是一种能引起雏鹅和雏番鸭发生急性或亚急性败血症的传染病,主要感染3-20日龄的雏鹅或雏番鸭。患病禽主要表现为全身败血性病变,局灶型肝炎、心肌炎和栓塞性肠炎。剖检变化主要在肠道的空肠和回肠,形成卡他性、纤维素性、坏死性肠炎,肠道形成栓塞。该病流行面广,传播速度快、发病率和死亡率较高,给我国水禽养殖业造成很大的经济损失。为深入研究小鹅瘟病毒的复制机理,了解其在宿主细胞内的定植和复制机制,本实验室开展了GPV与细胞蛋白互作研究。构建了鸭胚成纤维细胞酵母双杂交文库,利用酵母双杂交技术以GPV的结构蛋白VP1为诱饵,筛选出3个阳性克隆质粒。其中,一个基因长度为593bp,经测序Blast在线比对,该基因与原鸡属的翻译延伸因子EEF1A1蛋白同源性高达96%。为了进一步验证EEF1A1蛋白与VP1的相互作用,研究该蛋白对GPV增殖的影响,开展了本实验。构建原核表达质粒pET28a-EEF1A1(含His标签),进行原核诱导表达,SDS-PAGE分析,再用His标签树脂进行纯化。结果表明,纯化后的蛋白大小约为25 kDa。将实验室构建的pGEX4T-1-VP1进行原核诱导表达,表达的VP1蛋白大小约为108 kDa。采用GST-Pulldown蛋白互作技术,使EEF1A1与VP1形成了蛋白复合物,证明了在细胞外EEF1A1蛋白能与VP1蛋白结合。将原核诱导表达并纯化后的EEF1A1蛋白与GPV在细胞外感作,感染鸭胚成纤维细胞(DEF)作为实验组,以单独GPV感染DEF作为对照组,24 h后用荧光定量PCR方法检测GPV的核酸复制情况。结果表明,对照组GPV核酸复制拷贝数分别是实验组的2.3倍、5.4倍、7.7倍。由此说明,在细胞外,EEF1A1影响了小鹅瘟病毒在鸭胚成纤维细胞细胞表面的吸附,抑制了病毒的增殖。将pcDNA3.0-EEF1A1转染到DEF中,同时GPV感染DEF,作用12 h和24 h,分别设置单独GPV感染DEF细胞、GPV与空质粒pcDNA3.0、GPV与脂质体和GPV与高压灭菌水作为对照组,用荧光定量PCR检测GPV的核酸。结果表明,转染12 h时,实验组与对照组检测的GPV增殖含量差别不明显;转染24 h时,实验组GPV的核酸拷贝量是单独GPV感染DEF含量的2.7倍。由此说明,在细胞内,EEF1A1表达量的增加促进了GPV的增殖。本研究结果为鹅细小病毒复制机制研究提供了有益资料。
[Abstract]:Goose parvovirus (Goose parvovirus,GPV) disease, also known as goose plague, is an infectious disease that can cause acute or subacute septicemia in geese and muscovy ducks. It is mainly infected with 3-20-day-old geese or muscovy ducks. The main manifestations of diseased poultry are systemic septicemia, focal hepatitis, myocarditis and embolic enteritis. The changes were mainly in the jejunum and ileum of the intestinal tract, forming Carthamia, cellulosic, necrotizing enteritis and intestinal embolism. The epidemic area is wide, the spread speed is fast, the morbidity and mortality are relatively high, which causes great economic losses to the waterfowl breeding industry in our country. In order to study the replication mechanism of Gosling plague virus and understand its colonization and replication mechanism in host cells, the interaction between GPV and cell protein was studied in our laboratory. A yeast two-hybrid library of duck embryo fibroblasts was constructed and three positive clones were screened by yeast two-hybrid technique using GPV structural protein VP1 as bait. The length of one gene was 593 BP. The sequence Blast showed that the gene shared 96% homology with the translation extension factor EEF1A1 protein of the original genus. In order to further verify the interaction between EEF1A1 protein and VP1, and to study the effect of the protein on the proliferation of GPV, the experiment was carried out. The prokaryotic expression plasmid pET28a-EEF1A1 (containing His tag) was constructed and expressed in E. coli, analyzed by SDS-PAGE, and purified with His tag resin. The results showed that the purified protein was about 25 kDa. in size. The pGEX4T-1-VP1 constructed in the laboratory was induced to express in E. coli, and the size of the expressed VP1 protein was about 108 kDa.. GST-Pulldown protein interaction technique was used to form a protein complex between EEF1A1 and VP1, which proved that EEF1A1 protein could bind to VP1 protein. The prokaryotic expression of EEF1A1 protein was induced and purified in vitro with GPV in vitro. Duck embryo fibroblasts were infected with (DEF) as experimental group and GPV infected with DEF as control group. 24 h later, the nucleic acid replication of GPV was detected by fluorescence quantitative PCR (FQ-PCR). The results showed that the copy numbers of GPV DNA in the control group were 2.3 times, 5.4 times and 7.7 times as much as those in the experimental group, respectively. It is concluded that EEF1A1 affects the adsorption of Gosling plague virus on the surface of duck embryo fibroblasts and inhibits the proliferation of the virus. PcDNA3.0-EEF1A1 was transfected into DEF. At the same time, GPV infected DEF cells for 12 h and 24 h, respectively. DEF cells were infected with GPV alone, GPV and empty plasmid pcDNA3.0,GPV, liposome, GPV and high pressure sterilized water were used as control group. The nucleic acid of GPV was detected by fluorescence quantitative PCR. The results showed that there was no significant difference in the proliferation of GPV between the experimental group and the control group at 12 h after transfection, and the nucleic acid copy amount of GPV in the experimental group was 2.7 times as much as that of DEF infected with GPV alone at 24 h after transfection. Therefore, the increased expression of EEF1A1 promoted the proliferation of GPV in the cells. The results of this study provide useful information for the study of the replication mechanism of goose parvovirus.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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