蛋白质二硫键异构酶（PDI）表达与猪瘟病毒感染相关性研究

发布时间：2018-06-18 20:42

  本文选题：猪瘟病毒石门株 + PDI　； 参考：《西北农林科技大学》2015年硕士论文

【摘要】：猪瘟是由猪瘟病毒(Classical swine fever virus,CSFV)引起的猪的一种传染病,严重威胁到养猪业的发展,被世界动物卫生组织列为必须报告的动物疫病,我国将其列为一类动物疫病。我国研制出的猪瘟兔化弱毒疫苗虽然在猪瘟的预防控制方面起了很大的作用,然而近年来慢性猪瘟和非典型猪瘟不断发生,给养殖业造成了很大经济损失。CSFV致病机理的阐明将有助于猪瘟的防控。蛋白二硫键异构酶(protein disulphide isomerase,PDI)是一种分子伴侣,已经证明其在囊膜病毒侵入宿主细胞的过程中有重要作用,并且在内质网应激中也扮演重要角色。但PDI与CSFV的侵染机制是否有相关性,目前知之甚少。本研究建立了PDI实时荧光定量PCR检测方法,实现CSFV感染后,宿主组织及细胞水平的PDI表达检测。并在巨噬细胞中构建载体采用PDI过表达技术,研究分析CSFV与宿主细胞PDI之间可能的相互作用。(1)设计猪组织及细胞系PDI基因特异性扩增引物,利用RT-PCR扩增PDI基因,连接到pMD 19-T载体制备质粒标准品。通过优化反应条件,建立了检测猪PDI的SYBR GreenⅠ实时荧光定量PCR方法。该方法特异性强,敏感性高,重复性好。用该方法检测猪瘟病毒石门株(CSFV Shimen)感染和未感染的猪组织中PDI的表达量。结果显示,受CSFV感染后猪心脏、肝脏、脾脏、肺脏、肾脏和肠系膜淋巴结组织中的PDI表达量下降。(2)CSFV Shimen株感染巨噬细胞0h、12h、24h、48h后,提取不同时间点的细胞总RNA并收取细胞蛋白样品。采用荧光定量PCR方法检测巨噬细胞中的PDI基因的转录变化,采用western blot和免疫荧光法检测PDI蛋白表达变化,与对照组分析比较,CSFV Shimen株感染后,巨噬细胞PDI的转录和蛋白表达受到抑制。(3)针对PDI序列的编码区设计表达引物,扩增PDI序列。将其连接到pCDH-CMV-MCS-EF1-GreenPuro空载体上,构建了表达PDI蛋白的重组质粒,对重组质粒进行PCR、酶切和测序鉴定之后,证明重组质粒构建成功。将构建好的重组质粒转染进巨噬细胞,然后感染CSFV Shimen株。结果表明CSFV与PDI之间存在相互调控作用,CSFV可通过抑制PDI表达,进一步抑制ATF4基因的表达,而过表达PDI可使CSFV在巨噬细胞中的增殖受到抑制。综上所述,本研究建立了猪组织及细胞系的PDI荧光定量PCR方法,结合过表达基因技术,在CSFV感染的体内及体外实验获得了一致性的结果,CSFV感染可致宿主PDI表达下调,从而抑制ATF4基因的表达。同时,PDI在体内的过表达也对CSFV的复制具有调控作用对其增殖产生影响。
[Abstract]:Swine fever is an infectious disease of pigs caused by classical swine fever virus (CSFV). It is a serious threat to the development of pig industry. It is listed as an animal disease that must be reported by the World Organization of Animal Health (OIE). Although the attenuated swine fever rabbit vaccine developed in China has played a great role in the prevention and control of swine fever, chronic swine fever and atypical swine fever continue to occur in recent years. The explanation of the pathogenic mechanism of CSFV will contribute to the prevention and control of swine fever. Protein disulfide isomerase- (PDI) is a molecular chaperone, which has been proved to play an important role in the invasion of cysts into host cells, and also plays an important role in endoplasmic reticulum stress. However, little is known about the correlation between PDI and CSFV infection mechanism. In this study, a real-time fluorescent quantitative PCR assay was established to detect the expression of PDI in host tissues and cells after CSFV infection. PDI overexpression technique was used to construct the vector in macrophages. The possible interaction between CSFV and host cell PDI was studied. The PDI gene specific primers were designed for porcine tissue and cell line PDI gene amplification, and the PDI gene was amplified by RT-PCR. The plasmid standard was prepared by ligation to pMD 19 T vector. A real-time fluorescent quantitative PCR method for detection of porcine PDI by SYBR Green 鈪，

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