猪流行性腹泻病毒（PEDV）S基因功能域与其细胞适应性的关系
发布时间：2018-09-03 08:37
【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是猪的一种急性、高度传染性的肠道疾病。它的病原是猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PED V)。近几年,该病在中国广泛蔓延,造成了严重的经济损失,引起了中国各研究单位对其病原PEDV的关注。PEDV野外分离的流行毒株不易在实验室传代培养,对于该病的疫苗研发工作是一个很大的阻碍。本研究旨在通过靶向RNA重组反向操作平台,对影响PEDV在VERO上的细胞适应能力的S基因进行分段替换,最终找到该基因影响细胞适应能力的决定性功能域。本研究分为两个部分。第一部分:将实验室已经构建好的三个携带嵌合S基因的转移载体体外转录成RNA,通过电转,使嵌合的S基因整合进入PEDV病毒基因组,再将重组的PEDV(rPEDV)感染VERO细胞,从而获得能够在VERO细胞上产生CPE并且连续传代的重组PEDV。第一部分中嵌合的S基因分别是:1号,非细胞适应株(即野生型毒株,wild type,简称Wt)的S基因406-4162nt替换细胞适应毒株(即弱毒株,attenuated,简称Att)对应片段;2号,Wt的S基因1598-4162nt替换Att对应片段;3号,Wt的S基因406-1597nt替换Att对应片段。此外,在转移载体中,由R]LUC(海肾荧光素酶,RenillaLuciferase)基因替换了原本的ORF3基因。RLUC能够编码海肾荧光素酶蛋白,该蛋白与特定的底物相互作用后可以发出仪器能够检测的荧光,因此将其作为病毒细胞内增殖的标记基因。整个拯救过程重复三次,其中两次都成功拯救出了 3号重组病毒(Wt-S406-1597nt替换Att对应片段)。通过对拯救出的3号重组病毒部分代次的序列鉴定和RLUC发光值测定,我们发现2个平行实验组的S蛋白分别有一个氛基酸位点发生了突变,同时表现出不同的RLUC活性。通过以上结果,我们推测对于PEDV在VERO上的细胞适应能力,406-1597nt这一区段不起决定性作用。第二部分:构建新的嵌合S基因,连接跟第一部分相同的载体,获得新的转移载体。在第一部分实验结果的基础上,分别替换S基因1-405nt,1598-2820nt,2821-4162nt。三个转移载体分别命名为a,b,c。a为用非细胞适应株(即野生型毒株,wild type,简称Wt)的S基因1-405nt替换细胞适应毒株(即弱毒株,attenuated,简称Att)对应片段构建而成的质粒;b是Wt的S基因2821-4162 nt替换Att对应片段构建而成的质粒;c是用Wt的S基因1598-2820nt替换Att对应片段构建而成的质粒。通过对载体和S基因上的限制性单酶切位点的分析,最终选择位于1a基因9nt的Sma I,S基因405nt的Apa I和4154nt的PmlI。通过PCR分别扩增Wt毒株和Att毒株的小片段,再通过融合PCR,将小片段融合成两段覆盖酶切位点的大片段。将融合得到的大片段连接pJET-Blunt载体进行克隆并且测序。选择测序正确的Blunt载体,用对应的限制性内切酶将目的片段从Blunt载体上切下来。与此同时,用对应的限制性内切酶切开pPEDV载体。将切开的大小正确的条带胶回收纯化后,利用T4连接酶将目的片段和pPEDV载体连接,并且克隆测序,从而获得正确的转移载体。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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