猪传染性胃肠炎病毒M蛋白与宿主细胞相互作用蛋白的筛选鉴定

发布时间：2018-03-13 17:07

本文选题：猪传染性胃肠炎病毒(TGEV)　切入点：M蛋白　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：猪传染性胃肠炎(Porcine transmissible gastroenteritis,TGE)是由猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)引起的一类猪高度传染性疾病,尤其是冬季和早春寒冷季节,呈地方性暴发流行,临床症状主要以严重腹泻、呕吐及脱水为特征,不同年龄及品种的猪均可感染,尤其是2周龄以内哺乳仔猪,死亡率达100%。TGEV灭活和减毒活疫苗在亚洲已广泛应用于生产,但近年来在已免疫疫苗的猪场仍有致病性TGEV暴发流行,因此有必要研制有效的疫苗及开展病毒致病机制相关研究。病毒-宿主相互作用的研究可鉴定出致病基因,通过修饰或缺失致病基因来减弱病毒的毒力,有助于候选疫苗和抗病毒药物的研制以防控TGEV的感染。膜蛋白(M)是TGEV重要结构蛋白,主要包埋在病毒脂质囊膜中,是病毒装配期间构成冠状病毒颗粒的重要组成部分。同时,M蛋白高度保守,其在TGEV的致病性和病毒复制及转录过程中也发挥重要作用。M蛋白与S、N及E蛋白在内质网-高尔基中间体相互作用,促进病毒粒子的装配和出芽。M-M蛋白相互作用形成同源低聚体,是冠状病毒囊膜形成的基础,膜结合同样需要高效的M-M相互作用。但M蛋白与宿主蛋白的相互作用及其对TGEV感染的调控过程仍未知。为此本研究以TGEV M蛋白为研究对象,采用酵母双杂交系统筛选宿主细胞猪小肠上皮细胞(pIECs)与M蛋白互作的宿主蛋白;GST pull-down技术验证候选蛋白与M蛋白的互作。主要研究内容如下:(1)猪小肠上皮细胞(p IECs)cDNA文库的构建及鉴定提取p IECs的细胞总RNA,SMART法反转录合成cDNA,去除短片段后定与酵母文库表达载体pGADT7共转化至酵母菌株Y187,成功构建酵母双杂交cDNA文库。对文库质量进行检测,酵母文库滴度为5.0×107 cfu/m L,插入片段在500-2000 bp。结果表明,本试验成功构建了p IECs酵母双杂交cDNA文库,且文库质量较高,可用于M蛋白互作蛋白的筛选。(2)TGEV M蛋白诱饵质粒的构建及鉴定以本实验室构建并保存的质粒pFastBacTMDUAL-M为模板,聚合酶链反应(PCR)扩增M基因全长序列,然后定向克隆至酵母诱饵表达载体pGBKT7,转化至酵母菌株Y2HGold感受态细胞。对构建的诱饵载体pGBKT7-M进行细胞毒性、自转录激活活性及其在酵母细胞内的表达情况的检测。结果表明,空载体pGBKT7和诱饵载体pGBKT7-M的菌落大小和数量基本一致,构建的诱饵载体无细胞毒性;诱饵载体pGBKT7-M在SD/-Trp/X-α-Gal/AbA营养缺陷培养基上没有菌落生长,说明无自激活活性;Western blot结果可见蛋白条带约为50 kDa的融合蛋白,说明诱饵载体在酵母细胞Y2HGold中正确表达。构建的诱饵载体pGBKT7-M可用于p IECs酵母双杂交cDNA文库互作蛋白的筛选。(3)酵母双杂交筛选与M蛋白相互作用的蛋白根据Clontech公司Matchmaker?Gold Yeast Two-Hybrid System操作手册,以TGEV M基因构建酵母诱饵质粒pGBKT7-M,与p IECs酵母双杂交c DNA文库进行双杂交筛选,SD/-Trp/-Leu/-Ade、SD/-Trp/-Leu/-Ade/-His、SD/-Trp/-Leu/-Ade/-His/X-α-Gal/AbA营养缺陷培养基上三轮筛选,并对筛选出的蛋白进行Blast分析。最终筛选获得7种可能与M蛋白互作的宿主蛋白。其中,EIF4A2蛋白的重复性较高,且根据现有文献表明,EIF4A2参与促进病毒增殖及病毒蛋白合成。因此EIF4A2将作为候选蛋白进行下一步研究及验证。(4)GST pull-down实验验证M蛋白与候选蛋白的相互作用本部分利用GST pull-down实验验证M蛋白与候选蛋白EIF4A2的相互作用。构建原核表达载体pGEX-4T-M融合表达GST-M蛋白,Glutathione Sepharose 4B琼脂糖珠进行纯化;将EIF4A2基因片段连接到原核表达载体pET-28a构建重组表达载体pET-EIF4A2,Ni亲和层析柱对融合蛋白进行纯化。然后将GST-M融合蛋白作为诱饵蛋白固化在谷胱甘肽亲和树脂上,再用纯化后的His-EIF4A2蛋白溶液过柱,通过蛋白间的互作捕获候选蛋白,反复洗脱后经SDS-PAGE,Western blot检测M蛋白与候选蛋白EIF4A2间的互作。结果表明,M蛋白与候选蛋白EIF4A2确实存在特异性的相互作用。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

【参考文献】