鴨胚成纖維細胞翻譯延伸因子對鵝細小病毒增殖的影響
發(fā)布時間：2019-04-29 11:32
【摘要】：鵝細小病毒(Goose parvovirus,GPV)病,又稱小鵝瘟,是一種能引起雛鵝和雛番鴨發(fā)生急性或亞急性敗血癥的傳染病,主要感染3-20日齡的雛鵝或雛番鴨�；疾∏葜饕憩F(xiàn)為全身敗血性病變,局灶型肝炎、心肌炎和栓塞性腸炎。剖檢變化主要在腸道的空腸和回腸,形成卡他性、纖維素性、壞死性腸炎,腸道形成栓塞。該病流行面廣,傳播速度快、發(fā)病率和死亡率較高,給我國水禽養(yǎng)殖業(yè)造成很大的經(jīng)濟損失。為深入研究小鵝瘟病毒的復制機理,了解其在宿主細胞內(nèi)的定植和復制機制,本實驗室開展了GPV與細胞蛋白互作研究。構(gòu)建了鴨胚成纖維細胞酵母雙雜交文庫,利用酵母雙雜交技術(shù)以GPV的結(jié)構(gòu)蛋白VP1為誘餌,篩選出3個陽性克隆質(zhì)粒。其中,一個基因長度為593bp,經(jīng)測序Blast在線比對,該基因與原雞屬的翻譯延伸因子EEF1A1蛋白同源性高達96%。為了進一步驗證EEF1A1蛋白與VP1的相互作用,研究該蛋白對GPV增殖的影響,開展了本實驗。構(gòu)建原核表達質(zhì)粒pET28a-EEF1A1(含His標簽),進行原核誘導表達,SDS-PAGE分析,再用His標簽樹脂進行純化。結(jié)果表明,純化后的蛋白大小約為25 kDa。將實驗室構(gòu)建的pGEX4T-1-VP1進行原核誘導表達,表達的VP1蛋白大小約為108 kDa。采用GST-Pulldown蛋白互作技術(shù),使EEF1A1與VP1形成了蛋白復合物,證明了在細胞外EEF1A1蛋白能與VP1蛋白結(jié)合。將原核誘導表達并純化后的EEF1A1蛋白與GPV在細胞外感作,感染鴨胚成纖維細胞(DEF)作為實驗組,以單獨GPV感染DEF作為對照組,24 h后用熒光定量PCR方法檢測GPV的核酸復制情況。結(jié)果表明,對照組GPV核酸復制拷貝數(shù)分別是實驗組的2.3倍、5.4倍、7.7倍。由此說明,在細胞外,EEF1A1影響了小鵝瘟病毒在鴨胚成纖維細胞細胞表面的吸附,抑制了病毒的增殖。將pcDNA3.0-EEF1A1轉(zhuǎn)染到DEF中,同時GPV感染DEF,作用12 h和24 h,分別設(shè)置單獨GPV感染DEF細胞、GPV與空質(zhì)粒pcDNA3.0、GPV與脂質(zhì)體和GPV與高壓滅菌水作為對照組,用熒光定量PCR檢測GPV的核酸。結(jié)果表明,轉(zhuǎn)染12 h時,實驗組與對照組檢測的GPV增殖含量差別不明顯;轉(zhuǎn)染24 h時,實驗組GPV的核酸拷貝量是單獨GPV感染DEF含量的2.7倍。由此說明,在細胞內(nèi),EEF1A1表達量的增加促進了GPV的增殖。本研究結(jié)果為鵝細小病毒復制機制研究提供了有益資料。
[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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.3
本文編號：2468215
[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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.3
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