本文選題：豬瘟病毒石門株 + PDI　； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：豬瘟是由豬瘟病毒(Classical swine fever virus,CSFV)引起的豬的一種傳染病,嚴(yán)重威脅到養(yǎng)豬業(yè)的發(fā)展,被世界動(dòng)物衛(wèi)生組織列為必須報(bào)告的動(dòng)物疫病,我國將其列為一類動(dòng)物疫病。我國研制出的豬瘟兔化弱毒疫苗雖然在豬瘟的預(yù)防控制方面起了很大的作用,然而近年來慢性豬瘟和非典型豬瘟不斷發(fā)生,給養(yǎng)殖業(yè)造成了很大經(jīng)濟(jì)損失。CSFV致病機(jī)理的闡明將有助于豬瘟的防控。蛋白二硫鍵異構(gòu)酶(protein disulphide isomerase,PDI)是一種分子伴侶,已經(jīng)證明其在囊膜病毒侵入宿主細(xì)胞的過程中有重要作用,并且在內(nèi)質(zhì)網(wǎng)應(yīng)激中也扮演重要角色。但PDI與CSFV的侵染機(jī)制是否有相關(guān)性,目前知之甚少。本研究建立了PDI實(shí)時(shí)熒光定量PCR檢測方法,實(shí)現(xiàn)CSFV感染后,宿主組織及細(xì)胞水平的PDI表達(dá)檢測。并在巨噬細(xì)胞中構(gòu)建載體采用PDI過表達(dá)技術(shù),研究分析CSFV與宿主細(xì)胞PDI之間可能的相互作用。(1)設(shè)計(jì)豬組織及細(xì)胞系PDI基因特異性擴(kuò)增引物,利用RT-PCR擴(kuò)增PDI基因,連接到pMD 19-T載體制備質(zhì)粒標(biāo)準(zhǔn)品。通過優(yōu)化反應(yīng)條件,建立了檢測豬PDI的SYBR GreenⅠ實(shí)時(shí)熒光定量PCR方法。該方法特異性強(qiáng),敏感性高,重復(fù)性好。用該方法檢測豬瘟病毒石門株(CSFV Shimen)感染和未感染的豬組織中PDI的表達(dá)量。結(jié)果顯示,受CSFV感染后豬心臟、肝臟、脾臟、肺臟、腎臟和腸系膜淋巴結(jié)組織中的PDI表達(dá)量下降。(2)CSFV Shimen株感染巨噬細(xì)胞0h、12h、24h、48h后,提取不同時(shí)間點(diǎn)的細(xì)胞總RNA并收取細(xì)胞蛋白樣品。采用熒光定量PCR方法檢測巨噬細(xì)胞中的PDI基因的轉(zhuǎn)錄變化,采用western blot和免疫熒光法檢測PDI蛋白表達(dá)變化,與對照組分析比較,CSFV Shimen株感染后,巨噬細(xì)胞PDI的轉(zhuǎn)錄和蛋白表達(dá)受到抑制。(3)針對PDI序列的編碼區(qū)設(shè)計(jì)表達(dá)引物,擴(kuò)增PDI序列。將其連接到pCDH-CMV-MCS-EF1-GreenPuro空載體上,構(gòu)建了表達(dá)PDI蛋白的重組質(zhì)粒,對重組質(zhì)粒進(jìn)行PCR、酶切和測序鑒定之后,證明重組質(zhì)粒構(gòu)建成功。將構(gòu)建好的重組質(zhì)粒轉(zhuǎn)染進(jìn)巨噬細(xì)胞,然后感染CSFV Shimen株。結(jié)果表明CSFV與PDI之間存在相互調(diào)控作用,CSFV可通過抑制PDI表達(dá),進(jìn)一步抑制ATF4基因的表達(dá),而過表達(dá)PDI可使CSFV在巨噬細(xì)胞中的增殖受到抑制。綜上所述,本研究建立了豬組織及細(xì)胞系的PDI熒光定量PCR方法,結(jié)合過表達(dá)基因技術(shù),在CSFV感染的體內(nèi)及體外實(shí)驗(yàn)獲得了一致性的結(jié)果,CSFV感染可致宿主PDI表達(dá)下調(diào),從而抑制ATF4基因的表達(dá)。同時(shí),PDI在體內(nèi)的過表達(dá)也對CSFV的復(fù)制具有調(diào)控作用對其增殖產(chǎn)生影響。
[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，

本文編號：2036805