PRV EPO基因酵母双杂交诱饵质粒与PK-15细胞cDNA文库的构建

发布时间：2018-03-19 23:22

  本文选题：伪狂犬病病毒(PRV)　切入点：EPO　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：伪狂犬病(Pseudorabies,PR)是由伪狂犬病病毒(Pseudorabies Virus,PRV)引起多种家畜与野生动物以发热,奇痒,脑脊髓炎,流产及死亡为主要特征的急性传染病。猪是该病的贮存宿主和主要传染源,猪感染PRV后常表现为哺乳仔猪死亡、育肥猪生长停滞及妊娠母猪繁殖障碍。该病在欧洲、亚洲等多个国家呈蔓延趋势,已给世界养猪业带来了巨大的经济损失,中国尤为严重。PRV属于双链DNA病毒,大小约150 Kb,可编码70-100种蛋白质,其中EP0蛋白是PRV重要的早期蛋白。EP0蛋白包含与锌指结构类似的环指结构域,该结构域主要是结合DNA、RNA及蛋白质之间相互作用的区域,推测它在病毒与宿主相互作用方面具有重要意义。本研究构建了 pGBKT7-EP0诱饵质粒和PK-15细胞cDNA文库,为更好的研究PRV EP0蛋白与宿主细胞PK-15之间的相互作用机制提供基础。具体研究内容如下:第一部分:构建pGBKT7-EP0诱饵质粒采用PCR技术,扩增获得PRVEP0基因,将其克隆至酵母双杂交载体pGBKT7中,获得重组诱饵质粒pGBKT7-EP0,通过限制性内切酶EcoRI与Pst I双酶切鉴定与基因测序结果分析,表明成功构建诱饵质粒pGBKT7-EP0。将该质粒转入到Y2HGold酵母感受态细胞中,对其进行毒性检测,自激活效应检测,PCR以及Western-blot检测,结果表明诱饵质粒表达的产物对酵母细胞无毒性作用,无自激活效应,PCR和Western-blot检测结果证实诱饵质粒pGBKT7-EP0成功转入酵母细胞,并且能够表达融合蛋白 BD-EP0-Myc。第二部分:PK-15细胞cDNA文库构建及鉴定选取PRV的宿主细胞PK-15细胞来构建酵母双杂交cDNA文库。本试验从PK-15细胞提取总RNA,采用SMART和LD-PCR合成全长的ds cDNA,并对其进行均一化和SfiⅠ酶切处理。将得到的dscDNA连接到三框表达载体中。将这三个重组反应产物用电转化方法转入到HST08电转化感受态细胞中,构建含有三种读码框的初级文库,该文库库容分别为3.0X 106、2.0×106和2.0×106 CFU,插入片段均在500bp以上,阳性重组率均大于93%。再将三框初级文库共同电转化至感受态细胞HST08中进行扩增,提取质粒文库。最后将质粒文库转化到Y187酵母感受态细胞,得到的cDNA酵母文库总库容量为7.5× 105 CFU,基本符合酵母双杂交cDNA文库构建要求。综上,本课题成功构建了符合酵母双杂交筛选要求的诱饵质粒pGBKT7-EP0和酵母cDNA文库,为进一步研究PRV EPO蛋白与PK-15细胞蛋白之间的相互作用奠定了基础。
[Abstract]:Pseudorabies PRV (pseudorabies virus) is an acute infectious disease caused by pseudorabies virus (PRV) in many domestic and wild animals, characterized by fever, itchiness, encephalomyelitis, abortion and death. After infection with PRV, pigs often die of lactation, growth stagnation in fattening pigs and reproductive barriers in pregnant sows. The disease is spreading in many countries such as Europe and Asia, and has brought huge economic losses to the world pig industry. China is especially serious. PRV belongs to double-stranded DNA virus, about 150kb in size, and can encode 70-100 proteins. Among them, EP0 protein is an important early stage protein of PRV. EP0 protein contains ring finger domain similar to zinc finger structure. This domain is mainly a domain of interaction between RNAs and proteins, which is supposed to play an important role in the interaction between virus and host. In this study, pGBKT7-EP0 bait plasmid and cDNA library of PK-15 cells were constructed. The main contents are as follows: the first part: construct pGBKT7-EP0 bait plasmid to amplify and obtain PRVEP0 gene by PCR technique. The recombinant bait plasmid pGBKT7-EP0 was obtained by cloning it into yeast two-hybrid vector pGBKT7. The recombinant bait plasmid pGBKT7-EP0 was identified by restriction endonuclease EcoRI and Pst I, and the results of gene sequencing were analyzed. The results showed that the bait plasmid pGBKT7-EP0 was successfully constructed. The plasmid was transferred into Y2HGold yeast competent cells for toxicity detection, self-activation effect detection and Western-blot detection. The results showed that the product expressed by the bait plasmid had no toxic effect on yeast cells. The results of PCR and Western-blot showed that the bait plasmid pGBKT7-EP0 was successfully transferred into yeast cells. And can express the fusion protein BD-EP0-Myc.part two: PK-15 cell cDNA library was constructed and identified to construct yeast two-hybrid cDNA library with selected host cells PK-15 cells. In this experiment, total RNAs were extracted from PK-15 cells, and SMART and LD-PCR were used to synthesize the full-length cDNA library. The dscDNA was homogenized and digested with Sfi 鈪，

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