【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是豬的一種急性、高度傳染性的腸道疾病。它的病原是豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PED V)。近幾年,該病在中國(guó)廣泛蔓延,造成了嚴(yán)重的經(jīng)濟(jì)損失,引起了中國(guó)各研究單位對(duì)其病原PEDV的關(guān)注。PEDV野外分離的流行毒株不易在實(shí)驗(yàn)室傳代培養(yǎng),對(duì)于該病的疫苗研發(fā)工作是一個(gè)很大的阻礙。本研究旨在通過(guò)靶向RNA重組反向操作平臺(tái),對(duì)影響PEDV在VERO上的細(xì)胞適應(yīng)能力的S基因進(jìn)行分段替換,最終找到該基因影響細(xì)胞適應(yīng)能力的決定性功能域。本研究分為兩個(gè)部分。第一部分:將實(shí)驗(yàn)室已經(jīng)構(gòu)建好的三個(gè)攜帶嵌合S基因的轉(zhuǎn)移載體體外轉(zhuǎn)錄成RNA,通過(guò)電轉(zhuǎn),使嵌合的S基因整合進(jìn)入PEDV病毒基因組,再將重組的PEDV(rPEDV)感染VERO細(xì)胞,從而獲得能夠在VERO細(xì)胞上產(chǎn)生CPE并且連續(xù)傳代的重組PEDV。第一部分中嵌合的S基因分別是:1號(hào),非細(xì)胞適應(yīng)株(即野生型毒株,wild type,簡(jiǎn)稱(chēng)Wt)的S基因406-4162nt替換細(xì)胞適應(yīng)毒株(即弱毒株,attenuated,簡(jiǎn)稱(chēng)Att)對(duì)應(yīng)片段;2號(hào),Wt的S基因1598-4162nt替換Att對(duì)應(yīng)片段;3號(hào),Wt的S基因406-1597nt替換Att對(duì)應(yīng)片段。此外,在轉(zhuǎn)移載體中,由R]LUC(海腎熒光素酶,RenillaLuciferase)基因替換了原本的ORF3基因。RLUC能夠編碼海腎熒光素酶蛋白,該蛋白與特定的底物相互作用后可以發(fā)出儀器能夠檢測(cè)的熒光,因此將其作為病毒細(xì)胞內(nèi)增殖的標(biāo)記基因。整個(gè)拯救過(guò)程重復(fù)三次,其中兩次都成功拯救出了 3號(hào)重組病毒(Wt-S406-1597nt替換Att對(duì)應(yīng)片段)。通過(guò)對(duì)拯救出的3號(hào)重組病毒部分代次的序列鑒定和RLUC發(fā)光值測(cè)定,我們發(fā)現(xiàn)2個(gè)平行實(shí)驗(yàn)組的S蛋白分別有一個(gè)氛基酸位點(diǎn)發(fā)生了突變,同時(shí)表現(xiàn)出不同的RLUC活性。通過(guò)以上結(jié)果,我們推測(cè)對(duì)于PEDV在VERO上的細(xì)胞適應(yīng)能力,406-1597nt這一區(qū)段不起決定性作用。第二部分:構(gòu)建新的嵌合S基因,連接跟第一部分相同的載體,獲得新的轉(zhuǎn)移載體。在第一部分實(shí)驗(yàn)結(jié)果的基礎(chǔ)上,分別替換S基因1-405nt,1598-2820nt,2821-4162nt。三個(gè)轉(zhuǎn)移載體分別命名為a,b,c。a為用非細(xì)胞適應(yīng)株(即野生型毒株,wild type,簡(jiǎn)稱(chēng)Wt)的S基因1-405nt替換細(xì)胞適應(yīng)毒株(即弱毒株,attenuated,簡(jiǎn)稱(chēng)Att)對(duì)應(yīng)片段構(gòu)建而成的質(zhì)粒;b是Wt的S基因2821-4162 nt替換Att對(duì)應(yīng)片段構(gòu)建而成的質(zhì)粒;c是用Wt的S基因1598-2820nt替換Att對(duì)應(yīng)片段構(gòu)建而成的質(zhì)粒。通過(guò)對(duì)載體和S基因上的限制性單酶切位點(diǎn)的分析,最終選擇位于1a基因9nt的Sma I,S基因405nt的Apa I和4154nt的PmlI。通過(guò)PCR分別擴(kuò)增Wt毒株和Att毒株的小片段,再通過(guò)融合PCR,將小片段融合成兩段覆蓋酶切位點(diǎn)的大片段。將融合得到的大片段連接pJET-Blunt載體進(jìn)行克隆并且測(cè)序。選擇測(cè)序正確的Blunt載體,用對(duì)應(yīng)的限制性?xún)?nèi)切酶將目的片段從Blunt載體上切下來(lái)。與此同時(shí),用對(duì)應(yīng)的限制性?xún)?nèi)切酶切開(kāi)pPEDV載體。將切開(kāi)的大小正確的條帶膠回收純化后,利用T4連接酶將目的片段和pPEDV載體連接,并且克隆測(cè)序,從而獲得正確的轉(zhuǎn)移載體。
[Abstract]:Porcine epidemic diarrhea (PED) is a * * acute and highly contagious intestinal disease of swine. Its pathogen is Porcine epidemic diarrhea virus (PED * V). In recent years, the disease has been widely spread in China, causing serious economic losses, which has caused the pathogens in China to study the disease. Concern. The epidemic strains of PEDV isolated in the field are not easy to be subcultured in the laboratory, which is a great obstacle to the development of vaccine for PEDV. This study is divided into two parts. The first part is to transcribe three chimeric S gene transfer vectors constructed in the laboratory into RNA in vitro, through which the chimeric S gene is integrated into the PEDV virus genome, and then the recombinant PEDV (rPEDV) is infected with VERO cells to obtain the ability to produce on VERO cells. The chimeric S gene in the first part of the recombinant PEDV is: 1, the S gene 406-4162nt of the non-cell adapted strain (wild type, Wt) replaces the corresponding fragment of the cell adapted strain (attenuated strain, Att); 2, the S gene 1598-4162nt of Wt replaces the corresponding fragment of Att; 3, the S gene of Wt replaces the corresponding fragment of Att. In addition, the ORF3 gene was replaced by the R] LUC (Renilla Luciferase) gene in the transfer vector. RLUC can encode the marine luciferase protein, which interacts with a specific substrate and emits fluorescence detectable by the instrument, so it can be used as a virus cell. The whole rescue process was repeated three times, and two of them succeeded in rescuing the recombinant virus 3 (Wt-S406-1597nt replacing Att corresponding fragment). By sequencing and RLUC luminescence assay, we found that the S protein of the two parallel experimental groups had an atmospheric acid site respectively. These results suggest that 406-1597nt does not play a decisive role in the cell adaptation of PEDV to VERO. Part 2: Constructing a new chimeric S gene, linking the same vector as part 1, and obtaining a new transfer vector. On the basis of this, three transfer vectors named a, b, C.A were constructed by substituting 1-405nt of S gene for 1-405nt of Wt, 1598-2820nt and 2821-4162nt respectively; B was constructed by substituting corresponding fragments of S gene 2821-4162 of Wt for 1-405nt of Wt. The plasmid was constructed by replacing the corresponding fragment of Att with nt, and the plasmid C was constructed by replacing the corresponding fragment of Att with S gene 1598-2820nt of Wt. By analyzing the restriction single digestion site of vector and S gene, the SMI located in 9nT of 1a gene, APA I of 405nt of S gene and PMI of 4154nt of AT gene were finally selected. The small fragments of the strain were fused into two large fragments covering the digestion site by PCR. The large fragments were cloned and sequenced by ligating the pJET-Blunt vector. Restriction endonuclease was used to cut the pPEDV vector. After recovering and purifying the strip adhesive with the correct size, the target fragment was linked to the pPEDV vector by T4 ligase, and the pPEDV vector was cloned and sequenced.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.65

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