本文選題：豬傳染性胃腸炎病毒(TGEV)　切入點：M蛋白　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：豬傳染性胃腸炎(Porcine transmissible gastroenteritis,TGE)是由豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus,TGEV)引起的一類豬高度傳染性疾病,尤其是冬季和早春寒冷季節(jié),呈地方性暴發(fā)流行,臨床癥狀主要以嚴(yán)重腹瀉、嘔吐及脫水為特征,不同年齡及品種的豬均可感染,尤其是2周齡以內(nèi)哺乳仔豬,死亡率達(dá)100%。TGEV滅活和減毒活疫苗在亞洲已廣泛應(yīng)用于生產(chǎn),但近年來在已免疫疫苗的豬場仍有致病性TGEV暴發(fā)流行,因此有必要研制有效的疫苗及開展病毒致病機制相關(guān)研究。病毒-宿主相互作用的研究可鑒定出致病基因,通過修飾或缺失致病基因來減弱病毒的毒力,有助于候選疫苗和抗病毒藥物的研制以防控TGEV的感染。膜蛋白(M)是TGEV重要結(jié)構(gòu)蛋白,主要包埋在病毒脂質(zhì)囊膜中,是病毒裝配期間構(gòu)成冠狀病毒顆粒的重要組成部分。同時,M蛋白高度保守,其在TGEV的致病性和病毒復(fù)制及轉(zhuǎn)錄過程中也發(fā)揮重要作用。M蛋白與S、N及E蛋白在內(nèi)質(zhì)網(wǎng)-高爾基中間體相互作用,促進(jìn)病毒粒子的裝配和出芽。M-M蛋白相互作用形成同源低聚體,是冠狀病毒囊膜形成的基礎(chǔ),膜結(jié)合同樣需要高效的M-M相互作用。但M蛋白與宿主蛋白的相互作用及其對TGEV感染的調(diào)控過程仍未知。為此本研究以TGEV M蛋白為研究對象,采用酵母雙雜交系統(tǒng)篩選宿主細(xì)胞豬小腸上皮細(xì)胞(pIECs)與M蛋白互作的宿主蛋白;GST pull-down技術(shù)驗證候選蛋白與M蛋白的互作。主要研究內(nèi)容如下:(1)豬小腸上皮細(xì)胞(p IECs)cDNA文庫的構(gòu)建及鑒定提取p IECs的細(xì)胞總RNA,SMART法反轉(zhuǎn)錄合成cDNA,去除短片段后定與酵母文庫表達(dá)載體pGADT7共轉(zhuǎn)化至酵母菌株Y187,成功構(gòu)建酵母雙雜交cDNA文庫。對文庫質(zhì)量進(jìn)行檢測,酵母文庫滴度為5.0×107 cfu/m L,插入片段在500-2000 bp。結(jié)果表明,本試驗成功構(gòu)建了p IECs酵母雙雜交cDNA文庫,且文庫質(zhì)量較高,可用于M蛋白互作蛋白的篩選。(2)TGEV M蛋白誘餌質(zhì)粒的構(gòu)建及鑒定以本實驗室構(gòu)建并保存的質(zhì)粒pFastBacTMDUAL-M為模板,聚合酶鏈反應(yīng)(PCR)擴增M基因全長序列,然后定向克隆至酵母誘餌表達(dá)載體pGBKT7,轉(zhuǎn)化至酵母菌株Y2HGold感受態(tài)細(xì)胞。對構(gòu)建的誘餌載體pGBKT7-M進(jìn)行細(xì)胞毒性、自轉(zhuǎn)錄激活活性及其在酵母細(xì)胞內(nèi)的表達(dá)情況的檢測。結(jié)果表明,空載體pGBKT7和誘餌載體pGBKT7-M的菌落大小和數(shù)量基本一致,構(gòu)建的誘餌載體無細(xì)胞毒性;誘餌載體pGBKT7-M在SD/-Trp/X-α-Gal/AbA營養(yǎng)缺陷培養(yǎng)基上沒有菌落生長,說明無自激活活性;Western blot結(jié)果可見蛋白條帶約為50 kDa的融合蛋白,說明誘餌載體在酵母細(xì)胞Y2HGold中正確表達(dá)。構(gòu)建的誘餌載體pGBKT7-M可用于p IECs酵母雙雜交cDNA文庫互作蛋白的篩選。(3)酵母雙雜交篩選與M蛋白相互作用的蛋白根據(jù)Clontech公司Matchmaker?Gold Yeast Two-Hybrid System操作手冊,以TGEV M基因構(gòu)建酵母誘餌質(zhì)粒pGBKT7-M,與p IECs酵母雙雜交c DNA文庫進(jìn)行雙雜交篩選,SD/-Trp/-Leu/-Ade、SD/-Trp/-Leu/-Ade/-His、SD/-Trp/-Leu/-Ade/-His/X-α-Gal/AbA營養(yǎng)缺陷培養(yǎng)基上三輪篩選,并對篩選出的蛋白進(jìn)行Blast分析。最終篩選獲得7種可能與M蛋白互作的宿主蛋白。其中,EIF4A2蛋白的重復(fù)性較高,且根據(jù)現(xiàn)有文獻(xiàn)表明,EIF4A2參與促進(jìn)病毒增殖及病毒蛋白合成。因此EIF4A2將作為候選蛋白進(jìn)行下一步研究及驗證。(4)GST pull-down實驗驗證M蛋白與候選蛋白的相互作用本部分利用GST pull-down實驗驗證M蛋白與候選蛋白EIF4A2的相互作用。構(gòu)建原核表達(dá)載體pGEX-4T-M融合表達(dá)GST-M蛋白,Glutathione Sepharose 4B瓊脂糖珠進(jìn)行純化;將EIF4A2基因片段連接到原核表達(dá)載體pET-28a構(gòu)建重組表達(dá)載體pET-EIF4A2,Ni親和層析柱對融合蛋白進(jìn)行純化。然后將GST-M融合蛋白作為誘餌蛋白固化在谷胱甘肽親和樹脂上,再用純化后的His-EIF4A2蛋白溶液過柱,通過蛋白間的互作捕獲候選蛋白,反復(fù)洗脫后經(jīng)SDS-PAGE,Western blot檢測M蛋白與候選蛋白EIF4A2間的互作。結(jié)果表明,M蛋白與候選蛋白EIF4A2確實存在特異性的相互作用。
[Abstract]:Transmissible gastroenteritis (Porcine transmissible, gastroenteritis, TGE) from porcine transmissible gastroenteritis virus (Transmissible gastroenteritis virus, TGEV) for a class of porcine contagious disease caused by cold, especially in winter and early spring season, a local outbreak of the flow line, the main clinical symptoms of severe diarrhea, vomiting and dehydration characteristics, different age and variety pigs can be infected, especially within 2 week old piglets, the mortality rate of 100%.TGEV inactivated and live attenuated vaccine in Asia has been widely used in production, but in recent years has been in the vaccine of pig are pathogenic TGEV outbreaks, so it is necessary to develop an effective vaccine and research of pathogenic mechanism. Study of virus host interactions can be identified virulence genes, to reduce the virulence of the virus through the modification or deletion of genes, contribute to the candidate vaccine and antiviral The development of drugs to prevent TGEV infection. The membrane protein TGEV (M) is an important structural protein, mainly embedded in the viral lipid membrane, is an important part of virus particles formed during assembly of coronavirus. At the same time, M protein is highly conserved, its pathogenicity and replication and transcription of TGEV in.M proteins play an important role with S, N and E protein in the endoplasmic reticulum and Golgi intermediates interaction, assembly and budding of.M-M protein promotes viral particles formed by the interaction of homologous oligomer, is the basis of coronavirus capsule formation, membrane binding interactions also need efficient M-M. But the interaction between M proteins and host proteins and the regulation of TGEV infection is still unknown. The purpose of this study is to TGEV M protein as the research object, using the yeast two hybrid system host cells of porcine intestinal epithelial cell (pIECs) interacting with M protein in host eggs White; interaction GST pull-down technology to verify the candidate protein and M protein. The main contents are as follows: (1) porcine small intestine epithelial cells (P IECs) IECs P cDNA library construction and identification of extraction of the total cellular RNA, SMART was reverse transcribed into cDNA, and the yeast expression library CO transformation vector pGADT7 to yeast strain Y187 remove the fixed short fragment, successfully constructed the yeast two hybrid cDNA library. To detect the quality of the library, library titer was 5 * 107 cfu/m L fragment in 500-2000 bp. results, we constructed yeast two hybrid cDNA Library of P IECs, and the high quality library, can be used for screening M protein protein interaction. (2) construction and identification of plasmid pFastBacTMDUAL-M in the laboratory construction and the preservation of the template TGEV M protein bait plasmid, polymerase chain reaction (PCR) amplification of the full-length sequence of M gene, and then cloned into yeast expression vector PGBKT7, transformed into the yeast strain Y2HGold competent cells. The cytotoxicity of the bait vector pGBKT7-M was constructed, the detection of transcriptional activity and its expression in yeast cells. The results show that the consistent colony size and quantity of plasmid pGBKT7 and bait vector pGBKT7-M, to construct the bait vector without cell toxicity; bait vector in the pGBKT7-M SD/-Trp/X- alpha -Gal/AbA auxotrophic medium no colony growth, indicating no self activation; Western blot showed that protein bands of approximately 50 kDa fusion protein, indicating the correct expression of bait vector in yeast cell Y2HGold. The bait vector pGBKT7-M constructed can be used for screening of yeast two hybrid cDNA Library of P IECs interacting proteins. (3) the yeast two hybrid screening and M interacting protein according to Clontech Matchmaker Gold Yeast Two-Hybrid? System manual, to Construction of TGEV M gene in yeast two hybrid bait plasmids pGBKT7-M, SD/-Trp/-Leu/-Ade, SD/-Trp/-Leu/-Ade/-His and P were IECs C in yeast two hybrid DNA library, based on three screening culture SD/-Trp/-Leu/-Ade/-His/X- alpha -Gal/AbA nutritional deficiencies, and the screened protein was analyzed by Blast. The final screened 7 possible M protein and host protein interaction among them. EIF4A2 protein, high reproducibility, and according to the existing literature shows that EIF4A2 involved in promoting proliferation and viral protein synthesis. So EIF4A2 as candidate protein and validation of the next step. (4) the interaction between GST M protein and pull-down experimental verification of this part of the use of the candidate protein interaction between GST M protein pull-down experiment and the candidate protein EIF4A2. To construct prokaryotic expression vector of pGEX-4T-M fusion protein expression of GST-M, Glutathione Sepharose 4B agarose beads Purification; connecting the EIF4A2 gene fragment into the prokaryotic expression vector pET-28a to construct recombinant expression vector pET-EIF4A2, the column of the fusion protein was purified by affinity Ni. Then the GST-M fusion protein as the bait protein solidified in glutathione affinity resin, and then the purified His-EIF4A2 protein solution by column chromatography, protein interactions capture candidate protein, after repeated elution by SDS-PAGE and Western blot to detect the M protein interaction between EIF4A2 and proteins. The results showed that the M protein and the candidate protein EIF4A2 exist specific interactions.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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