【作者】 孙东波；

【导师】 陈洪岩；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2008， 博士

【摘要】 猪流行性腹泻(porcine epidemic diarrhea, PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)引起猪的一种急性、高度接触性肠道传染病。在过去30年间,虽然PEDV常规疫苗被广泛应用,但是PED在各国猪场中的感染仍然非常严重,给养猪业带来巨大经济损失。所以,关于PEDV的新型疫苗、侵入机制和免疫机理的研究是十分必要的。众所周知,病毒的抗原表位、受体与受体结合域在病毒侵染和抗病毒免疫中起着重要作用。S蛋白是PEDV一个表面的结构蛋白,它既含有介导病毒侵入宿主细胞的受体结合域,又拥有介导机体产生病毒中和抗体的抗原表位。鉴于此,本研究对PEDV S蛋白的抗原表位和受体结合域进行筛选与鉴定,为PEDV诊断方法和新型疫苗的研究以及抗病毒免疫策略的设计提供信息。为获得PEDV S基因及其分子特性,本试验克隆了PEDV CV777毒株细胞适应毒的S基因,序列比对结果表明,获得的S基因与PEDV CV777毒株S基因参考序列核苷酸的同原性为99.4 %,推导氨基酸的同原性为99.8 %。S蛋白氨基酸的疏水性、信号肽序列和N-侧链连接糖基化位点分析表明,克隆的S基因保持了其亲本毒株CV777 S基因的分子特性。根据冠状病毒II群成员S蛋白S1和S2亚基之间的保守基序(GxCx和保守九肽),PEDV S蛋白可以被划分为2个功能区即S1(第1～789位氨基酸)和S2(第790～1383位氨基酸),其中S1区包含了病毒主要的中和表位和受体结合域。为了分析S蛋白S1区抗原表位特征,利用fd丝状噬菌体展示系统,构建了S1基因特异性肽库,以PEDV多克隆血清为靶蛋白对构建的肽库进行3轮生物淘选,结果获得了3个高亲和力序列,分别命名为S1P1(第248～280位氨基酸)、S1P2(第442～499位氨基酸)和S1P3(第697～742位氨基酸)。ELISA和Western blot结果显示,S1P1、S1P2和S1P3的GST融合蛋白均与PEDV多克隆血清反应,其中S1P3反应性最强。为了进一步揭示S1P1、S1P2和S1P3短肽的抗原性,三个短肽GST融合蛋白和它们串联后GST融合蛋白(S1P123-GST)的单因子鼠血清被制备。间接免疫荧光试验(IFA)和ELISA结果证实,抗S1P2、S1P3和S1P123 GST融合蛋白的单因子鼠血清能识别天然的PEDV。本试验结果表明,S1P1、S1P2和S1P3是PEDV S蛋白3个线性抗原表位,S1P2和S1P3表位具有良好的免疫原性。PEDV S蛋白S1区在介导中和抗体产生过程中发挥重要作用。为了鉴定S蛋白S1区的免疫优势区,利用PCR扩增了4个相互重叠、覆盖S1基因的片段,分别命名为S1A、S1B、S1C和S1D。四个片段PCR产物分别克隆到pGEX-6p-1原核表达载体后,经IPTG诱导均获得了表达。Western blot和ELISA结果显示,S1D-GST融合蛋白(第636～789位氨基酸)与PEDV的多克隆抗体具有强反应性。IFA和Western blot结果表明,抗S1D-GST融合蛋白的鼠血清能够识别天然的S蛋白。病毒中和试验表明,S1D-GST融合蛋白能介导鼠产生对PEDV具有中和作用的抗体。为了对中和表位区(S1D)的抗原表位进行精确定位,制备并获得了6株抗S1D特异性的单克隆抗体,Western blot结果显示,6株单克隆抗体均能识别天然的S蛋白。利用噬菌体展示和肽扫描技术对6株单克隆抗体识别的表位进行了鉴定,结果显示,单克隆抗体2C4、3C3和5F8识别的表位是S蛋白的第744～759位氨基酸(S1D5),单克隆抗体3G3、6E6和3G5识别的表位是S蛋白的第756～771位氨基酸(S1D6)。ELISA和IFA结果表明,抗S1D5和S1D6表位融合蛋白的鼠血清均能识别天然的S蛋白。进一步的肽扫描(Pepscan)结果显示,S1D5表位的核心序列是Y748SNIGVCK755(SS5),S1D6表位的核心序列是L764QDGQVKI771(SS6)。病毒细胞受体和受体结合域的信息能为病毒疫苗和抗病毒药物设计提供策略。为了揭示PEDV的受体结合域信息,本试验以PEDV可溶性的细胞受体猪氨基肽酶N(pAPN)为靶蛋白对S1基因特异性肽库进行3轮生物淘选,然后对30个淘选的噬菌体克隆进行测序。序列分析发现,2个噬菌体克隆展示无义氨基酸序列,其余28个噬菌体克隆展示的氨基酸序列位于S蛋白第249～529位氨基酸区域,这个区域被命名为MRR。为了进一步印证MRR区与pAPN在体外的相互作用,利用Bac-to-Bac杆状病毒表达系统对MRR基因进行真核表达。Western blot结果表明,表达的MRR重组蛋白能够与兔抗PEDV的多克隆抗体反应。但是,进一步的pull-down实验结果显示,利用抗pAPN的鼠血清通过Western blot检测不到与MRR重组蛋白结合的pAPN。本试验结果提示,PEDV S蛋白第249～529位氨基酸区域(MRR)是pAPN细胞受体的一个潜在的结合区域。本研究鉴定出PEDV S蛋白5个线性抗原表位即S1P1(第248～280位氨基酸)、S1P2(第442～499位氨基酸)、S1P3(第697～742位氨基酸)、SS5(第748～755位氨基酸)和SS6(第764～771位氨基酸),1个中和表位区(S1D,第636～789位氨基酸),1个猪氨基肽酶N潜在的受体结合域(MRR,第249～529位氨基酸)。这些抗原表位以及受体结合域的揭示对进一步分析PEDV S蛋白的结构与功能以及建立以表位为基础的抗原抗体诊断方法和基于表位的疫苗设计具有指导意义。更多还原

【Abstract】 Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV). During the past 30 years, in spite of preparation of inactivated and live attenuated vaccines, PED has frequently broken out in many swine-raising countries of Asia, resulting in large economic losses. Thus, there is a need to develop novel vaccine of the PEDV, and understand the mechanism of viral entry and immunization. The S protein of PEDV is a glycoprotein localized on the virion surface, which plays a key role in the induction of the neutralizing antibodies and specific receptor binding and cell membrane fusion, because of possessing the neutralizing epitopes and receptor binding domain. Given this, screening and identification of the receptor binding domain and antigenic epitopes were carried out in this study. It is necessary to provide some valuable information for development of diagnosis technology and novel vaccine, and for designing antiviral immunization strategy of PEDV.In order to obtain information about molecular properties of PEDV S gene, the S gene of PEDV strain CV777 adapted to Vero E6 cells was sequenced, using three overlapping cDNA clones which were amplified by RT-PCR based on viral genome RNA. Sequence analysis showed that identity of nucleotides and deduced amino acids was 99.4 % and 99.8 % respectively, comparing with the reference sequence of S gene of PEDV strain CV777. Analysis result of the amino acids hydrophobicity, signal peptide sequence and N-linked glycosylation demonstrated that these molecular properties of the cloned gene S were the same to the parental virus PEDV strain CV777. S protein was divided into the S1 domain (aa 1~789) and S2 domain (aa 790~1383) according to the presence of conserved nonamer and the GxCx motifs at the proteolytic cleavage site of S protein in other members of coronavirus, group II. The S1 domain contains major neutralization epitopes and receptor binding domain of PEDV.To analyze antigenic epitopes of PEDV S protein, the S1 gene targeted libraries containing the major immunodominant region S1 (aa 1~789) of PEDV spike glycoprotein gene were constructed using the fd phage display system which the exogenous polypeptides were expressed in the N-terminal of the fd phage gene III coat protein. The S1 libraries were panned three times with the purified rabbit sera against PEDV. Three short peptides which were displayed by recombinant fd phages showed strong binding affinity with the PEDV antisera, and were designated S1P1 (aa 248~280), S1P2 (aa 442~499) and S1P3 (aa 697~742) respectively. The results of ELISA and Western blot indicated that the GST fusion proteins of the three short peptides were all recognized by the PEDV antisera and S1P3 showed strong binding activity. To further determined antigenicity of the epitopes S1P1, S1P2 and S1P3, the antisera of the GST fusion proteins S1P1, S1P2, S1P3 and S1P123 (fused epitopes of S1P1, S1P2 and S1P3) were prepared. The result of the IFA and ELISA demonstrated that the epitopes S1P2, S1P3 and the fusion epitope S1P123 were able to induce the S1-specific antisera with the binding ability to the native S protein of PEDV cultured in Vero E6 cells. The result showed that the epitopes S1P1, S1P2 and S1P3 were three linear antigenic epitopes on the spike protein of PEDV, and the epitopes S1P2 and S1P3 had good immunogenicity.The S1 domain of PEDV S protein plays a key role in the induction of the neutralizing antibodies. To identify immunodominant region on S protein, the gene encoding its major immunodominant region S1 was amplified by PCR. Four truncated S1 proteins spanning the entire S1 domain fused to GST protein were prepared. These recombinant proteins were designated as S1A-GST, S1B-GST, S1C-GST and S1D-GST. To identify the most important antigenic region of the S1, the truncated S1-GST fusion proteins were examined for their ability to react with immune serum against PEDV and to elicit the formation of neutralization antibodies in immunized animals. It was found that the region S1D (aa 636~789) was able to react with PEDV antiserum and to elicit formation of neutralization antibodies in mice. Moreover, the immune serum against S1D showed the binding ability to the native S protein of PEDV. To accurately identify epitopes on neutralizing epitope region S1D, six specific monoclonal antibodies (McAbs) against the S1D region were prepared. In western blot, these McAbs could react with the native S protein of PEDV. In order to mapping epitopes of the domain S1D, the S1-phage library and Pepscan technology were carried out. The result showed that the McAbs 2C4, 3C3 and 5F8 recognized the epitope S1D5 (aa 744-759), and the McAbs 3G3, 6E6 and 3G5 recognized the epitope S1D6 (aa 756-771). The antisera of the epitopes S1D5 and S1D6 could react with the native S protein of PEDV. Furthermore, Pepscan of the two linear epitopes demonstrated that SS2 (Y748SNIGVCK755) and SS6 (L764QDGQVKI771) are two core epitopes on the epitopes S1D5 and S1D6, respectively.Information of cellular receptor and its receptor binding region could provide better strategies for developing effective vaccine and medicine. To identify the receptor binding domain of porcine aminopeptidase N (pAPN) receptor of PEDV, the S1 gene targeted libraries of PEDV were panned three times with the soluble pAPN based on standard procedure. Thirty phage clones which selected randomly from the binding-phages of pAPN, were sequenced by the dideoxy method. The result showed that polypeptides displayed by twenty-eight phage clones (excepted two meaningless sequences) were focused on 249~529 amino acids (MRR) on S protein of PEDV. In order to investigate interaction between pAPN and the region MRR of the PEDV S protein, His fusion gene of the region MRR was expressed in insect cells (sf9) using Bac-to-Bac? baculovirus expression system. In Western blot, MRR fusion protein with His tag could react with antiserium of PEDV. However, the further pull-down assay demonstrated that the binding activity of the recombinant protein MRR and pAPN could not be detected in Western blot. These data suggested that the region MRR of the PEDV S protein was a potential receptor binding domain of the porcine aminopeptidase N (pAPN).All together, five linear antigenic epitopes S1P1 (aa 248~280), S1P2 (aa 442~499), S1P3 (aa 697~742), SS5 (aa 748~755) and SS6 (aa 764~771), one neutralizing epitopes (S1D, aa 636~789), and one potential receptor binding domain (MRR, aa 249~529) of the porcine aminopeptidase N (pAPN) were identified on the S protein of PEDV. These data could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of porcine epidemic diarrhea virus and further structure and function analysis of spike protein.更多还原