【作者】 董波；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 基础兽医学， 2014， 博士

【摘要】 猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus，PHEV)是引起猪血凝性脑脊髓炎(porcine hemagglutinating encephalomyelitis)的致病原，属于β冠状病毒属的成员。PHEV的主要感染对象为哺乳期的仔猪。一旦病毒感染仔猪，其传播速度很快，且会造成很高的死亡率。PHEV与其它冠状病毒结构相似，包含了五种结构蛋白，即小膜蛋白(E蛋白)、膜蛋白(M蛋白)、刺突蛋白(S蛋白)、核衣壳蛋白(N蛋白)以及血凝素——酯酶蛋白(HE蛋白)。通过对其它冠状病毒的研究发现，S蛋白在病毒入侵和感染细胞过程中，起到了关键的作用。冠状病毒S蛋白分为S1和S2两个功能域，这两个功能域在S蛋白中发挥着各自的作用，其中S1功能域的主要功能是能够与易感细胞表面的受体结合，并能够诱导S2功能域与细胞膜的融合。S1功能域中包含着与细胞受体相结合的区域，即受体结合域(RBD)。而对于RBD的研究，则有助于更深层次的了解病毒入侵机制以及研发出更有针对性的病毒疫苗。本研究依据冠状病毒S蛋白划分标准，通过与部分冠状病毒S蛋白氨基酸序列进行比对，将PHEV S蛋白划分为S1和S2功能域，即第1-794位氨基酸被定义为是S1功能域，第795-1349位氨基酸被定义为S2功能域。将PHEV与其它冠状病毒的理化性质进行比较分析，以及对S蛋白亲水性参数，可及性参数，β-转角等进行预测后，证明了S蛋白具有较强的亲水能力，在S1功能域上存在着10个能够与受体发生结合的可及性片段。参照S蛋白的生物信息学分析结果，将S蛋白S1功能域分为三段基因片段，分别命名为：S1-291、S277-794和S548-868，把三段基因片段与原核表达载体pGEX-4T-1的线性化片段连接，构建重组表达载体pGEX-4T-1-S1-291，pGEX-4T-1-S548-868和pGEX-4T-1-S277-794，将重组表达载体分别转化入BL21感受态细胞中，经IPTG诱导，三段蛋白成功的以可溶性表达。在本课题组的前期工作中，已经发现NCAM能够与PHEV S蛋白S1功能域发生相互作用。并且，通过实验证实了NCAM参与了猪血型性脑脊髓病毒侵入神经细胞的过程，在PHEV侵染神经细胞过程中发挥着作用，其极有可能为PHEV在N2a细胞上的受体。因此，本研究将NCAM基因片段连接到pET28a表达载体，构建重组表达载体pET-NC，转化入BL21感受态细胞中，经IPTG诱导在大肠杆菌中获得了可溶性表达蛋白。为了找寻PHEV S1功能域上能够与NCAM蛋白发生相互作用的区域，本研究使用GST pull-down实验来筛选相互作用的片段，结果发现S277-794与NCAM存在着相互作用，而其它两段蛋白（S1-291和S548-868）没有这种结合能力。随后使用了酵母双杂交实验对结果进行验证，结果证实了S277-794能够与NCAM发生相互作用，而这种相互作用不存在于S1-291与NCAM以及S548-868与NCAM之间。然而，在S277-794与S1-291之间存在着相互重叠的部分，并且S277-794与S548-868之间也有相互重叠的部分。因此，依据这些结果，本研究预测出一段包含有258aa的小片段(S291-548)，这个小片段很有可能为PHEV S蛋白上介导与NCAM发生相互作用的关键区域，其很有可能为PHEV S蛋白上的RBD。为了对S291-548进行进一步的分析，本研究将S291-548进行大肠杆菌体外表达，获得了含有目的蛋白片段的融合蛋白。将S291-548与NCAM作用后的混合液与吸附在Glutathione-Sepharose4B颗粒上的S277-794-GST融合蛋白进行反应，使用抑制蛋白相互作用的GST pull-down实验来证明S291-548能够与NCAM的相互作用，结果表明，S291-548重组蛋白因为与NCAM发生了结合而抑制了S277-794-GST与NCAM的结合，证明了筛选出的S291-548能够与NCAM发生相互作用。同时，使用S291-548的重组表达蛋白免疫家兔，制备多克隆抗体，将多抗与病毒等体积混合后，接种到铺满单层的N2a细胞中，接毒48h后，分别将阴性对照组、实验组和阳性对照组的细胞培养液上清取出进行血凝实验，结果表明实验组HEV血凝价为1:22，而阳性对照组血凝价为1:24，两组间血凝价有明显差异。证明S291-548能够抑制PHEV对N2a细胞的感染。这些结果说明S291-548可能是NCAM的一个潜在的RBD，这一结果的获得对于进一步分析PHEV S蛋白的结构与功能以及设计更具有靶向性的疫苗奠定了基础。更多还原

【Abstract】 Porcine hemagglutinating encephalomyelitis coronavirus (PHEV) is a member ofthe Coronaviridae family, which causes porcine encephalomyelitis. PHEVpredominantly affects piglets. When PHEV infects the piglets, its transmission speedwill be quick and with a high mortality rate. The major structural proteins of the virusnamed small membrance(E), membrance(M),spike(S),nucleocapsid (N) protein andhemagglutinin-esterase (HE).Base on the study of other coronavirus, the Coronavirus Spike (S) protein isdivided into S1functional domain and S2functional domain.The S1functionaldomain initiates entry of viruses into cells by binding to cell surface receptors, and theS2functional domain helps viral fusion with cellular membranes. S1function domaincontains the receptor binding domain (RBD). Given this, it is necessary that screeningand identification of the RBD to provide some valuable information for developmentof diagnosis technology and novel vaccine of PHEV.According to the presence of conserved nonamer and the GxCx motifs at theproteolytic cleavage site of S protein in other members of coronavirus, PHEV Sprotein was divided into S1and S2function domain, The1-794aa of PHEV S proteinis the S1and the795-1349aa is the S2.Therefore, based on standard of coronavirus Sprotein, and compared with some other coronavirus S protein amino acid sequence, Sprotein is with strong hydrophilic ability, and there are10domains maybe combinewith the receptor accessibility on the S1.Considering to accessibility domains predicted, the S1functional domains ofPHEV S protein was divided into three truncated fusion proteins, S1-291、S277-794andS548-868.The plasmids pGEXS1-291, pGEXS277-794, pGEXS548-868were individuallytransformed in E. coli BL21cells separately and three truncated fusion proteins wereexpressed successfully.In the preliminary work of our project group, it has found thatthe neural cell adhesion molecule (NCAM) interacts with S1functional domain of PHEV S protein.And it was confirmed that NCAM participates in the process bywhich PHEV infects neurons.NACM may be a receptor for PHEV in N2acells.Therefore, pET-NC was transformed in E. coli BL21cells and the NCAM genewas expressed successfully,too.To screened the domains that could interact with NCAM,this study used GSTpull down and the interaction was further confirmed by a yeast wo-hybrid systemassay. The results showed that S277-794were identified to interact with NCAM,whileS1-291and S548-868not.S277-794is located in S1subunit. In addition,because S1-291orS548-868could not interact with NCAM, and there are overlapping region betweenS277-794and S1-291as well as S277-794and S548-868, we conclude that the S1subunit maycontain a minimal cellular receptor binding region. These findings suggested that the258-amino-acid fragment, residues291to548, may be the NCAM RBD of the Sprotein.Finally, a small fragment (258-amino-acid fragment, residues291to548) onthe PHEV S protein was posited to be the minimum number of amino acids necessaryto interact with NCAM, and it is very likely to the RBD that mediates PHEV bindingto NCAM.In order to further analysis for fragment, His fusion gene of the S291-548wasexpressed in BL21. The mixture contained S291-548and NCAM was incubated withS277-794-GST immobilized Glutathione Sepharose4B.The inhibiting protein interactionGST pull-down experiment shows that S291-548completely blocked the bindingbetween S277-794and NCAM，further proved that S291-548is able to interact with theNCAM.The polyclonal antibody of S291-548was prepared with immunized rabbit. Aftermixing the prepared polyclonal antibody and PHEV, and then was inoculated to theneural cells with the HEV. At48h after the inoculation, the hemagglutination titer ofthe HEV in the supernatant fluid of the cell culture was1:22test group,and thehemagglutination titer of HEV in the supernatant fluid of the cell culture was1:24inthe control group. A difference of two gradients existed between the hemagglutinationtiters of the two groups, thus the difference was significant.Results showed thatS291-548protein antibodies could inhibit the proliferation of PHEV in the cell. Theseresults provided evidences that S291-548may be a potential of NCAM receptor binding domain. Meanwhile,these data could provide the basis for the development ofidentifying the effective target epitope for a S protein-based vaccine and furtherstructure and function analysis of S protein.更多还原