【作者】 高巍；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 基础兽医学， 2010， 博士

【摘要】 猪血凝性脑脊髓炎(Porcine hemagglutinating encephalomyelitis)是由血凝性脑脊髓炎病毒(Hemagglutinating encephalomyelitis virus, HEV)引起仔猪的一种急性、高度接触性传染病。HEV属于冠状病毒属的成员,其主要侵害1～3周龄的仔猪,临床上以仔猪呕吐、衰竭和明显的神经症状为主要特征,死亡率高达20～100%。自1958年本病在加拿大的安大略省首次爆发以来,此后许多国家均有该病的报道。血清学检测结果证明猪感染HEV很普遍,可能呈世界性分布。2006年8月,阿根廷的部分猪场发生该病,导致1226头猪死亡,发病率高达52.6%。我国早在1985年报道了北京郊区某种猪场发生HEV感染,其后吉林、辽宁、山东以及台湾等地区也相继报道。1994年台湾地区发生HEV大规模流行,病死率几乎达100%,造成严重的经济损失。国外学者的血清学调查发现,猪的HEV感染非常普遍,呈世界性分布,对养猪业发展已构成严重威胁。但HEV是如何感染机体的,目前还不清楚,正是由于其感染机制不清楚,所以在该病的防治方面还缺乏有效的手段。鉴于该病的流行趋势和对我国猪群所造成的危害,积极开展HEV致病机制的研究具有重要的理论价值和现实意义。目前,对于HEV的致病机理尚不清楚。为了更好地防治该病,或者从根本上彻底消除该病,对其发病机理的研究显得尤为重要。由于冠状病毒具有识别特定宿主细胞的相应受体,继而选择性的感染特定物种细胞的特点,因此,在神经系统病变部位,尤其是神经细胞表面很可能存在HEV受体,也正是这些受体介导了病毒侵入细胞的途径。但是,HEV的细胞受体及由受体介导的感染机制目前还不清楚。为了研究猪血凝性脑脊髓炎病毒在神经细胞上的受体及由其介导的感染机制,本研究首先培养原代小鼠神经细胞,建立小鼠神经细胞T7噬菌体表面展示文库,利用真核表达的S1蛋白和HE蛋白分别对文库进行筛选,通过筛选共获得四种侯选受体基因,分别是神经细胞粘附分子、剪接因子3b亚基2、组氨酸脱乙酰基酶2和核糖体蛋白S13。用生物学软件对四种侯选受体基因进行结构和功能分析发现其中的三种基因在体内各种组织细胞中表达,推测其介导HEV嗜神经传播的可能性较小,未对其做深入鉴定;而神经细胞粘附分子(NCAM)的作用和HEV的嗜神经性密切相关,推测其可能与HEV病毒感染相关。对NCAM基因进行原核表达,制备其多克隆抗体。利用制备的NCAM蛋白多抗进行细胞阻断试验,表明NCAM蛋白的抗体可显著抑制HEV对小鼠神经细胞的感染,说明NCAM蛋白在介导HEV感染神经细胞中起重要作用。采用半定量RT-PCR方法检测NCAM蛋白在猪中枢神经系统中的表达量,NCAM蛋白在大脑、脊髓中表达量比其它组织大,这也与接种HEV后病毒在脑、脊髓中含量比其它组织多相符合,表明NCAM与HEV感染相关。采用免疫组化技术检测NCAM蛋白在中枢神经系统包括大脑、小脑和脊髓中均有表达,这与HEV在猪中枢神经系统的分布特征基本相同,进一步说明NCAM蛋白在HEV感染神经细胞中起重要作用,NCAM蛋白可能是HEV在神经细胞上的受体,为进一步研究HEV的致病机制,研究抗HEV的药物或疫苗奠定基础。此外,本研究对由病毒引起的慢性神经系统疾病也具有重要的参考价值。原代培养小鼠神经细胞。取出生后1d的新生小鼠,断颈处死后,用眼科镊剥出整个脑组织,用0.25 %胰蛋白酶消化处理后,置37℃、5 %CO2培养箱内培养。用倒置显微镜观察刚分离的神经细胞,细胞数量很多,呈圆形,有少量细胞长有短小的轴突。培养4～5d后,大部分细胞都可见轴突,并开始相互迁移靠近,互相形成网络。培养6～8 d后,细胞间网络更加密集,轮廓清晰,立体感强。培养8～10d后,细胞间开始形成网束状。13d～20d为生长高潮,生长趋于稳定,细胞成熟。随后细胞数减少,空泡变性细胞开始出现。研究发现原代培养13d到20d的小鼠神经细胞可以用于建立噬菌体文库。构建小鼠神经细胞T7噬菌体展示文库。用Trizol提取小鼠神经细胞总RNA,用Poly(A)Quick mRNA Isolation Kit提纯mRNA,然后分别使用Novagen公司的OrientExpress Random Primer cDNA Synthesis Kit、EcoRⅠ/ HindⅢEnd Modification Kit、Mini Column Fractionation Kit、DNA Ligation Kit和T7 Select 10-3b Cloning Kit,按照各试剂盒说明书进行操作,构建了体外培养的小鼠神经细胞T7噬菌体展示文库,未扩增文库的滴度为6.4×107 pfu/mL,文库容量为1.52×107个重组子。对随机挑取的100个噬菌斑进行PCR鉴定,文库重组率为94%,86%的插入片段大于0.3kb,扩增后文库滴度为9×1010pfu/mL。扩增后的文库可以用来筛选HEV的受体。血凝性脑脊髓炎病毒受体基因的筛选。小鼠神经细胞长成单层后,用真核表达的S和HE蛋白对扩增后的文库进行筛选,共进行5轮筛选,将第五轮筛选后的上清铺板。挑选单个噬菌斑,提取噬菌体DNA ,以提取的噬菌体DNA为模板,进行PCR扩增,检测电泳插入片段大小。每次筛选随机挑取100个噬菌斑进行PCR,结果显示80个以上的插入片段大小一致,占挑取的总噬菌斑数的80%以上。将筛选获得的噬菌体插入片段送生物公司测序。测序后,经BLAST分析,共筛选出四种不同的蛋白基因。但是只有神经细胞粘附分子(NCAM)是神经细胞相关性蛋白基因,其它三种蛋白基因在体内到处都有表达,不可能是HEV的受体,所以不对这三种蛋白进行分析。用DNAStar软件分析NCAM的核酸序列,用ScanProsite和InterPro Scan分析氨基酸序列。分析结果显示神经细胞粘附分子的作用和HEV的嗜神经性密切相关。NCAM基因的原核表达。为进一步研究NCAM蛋白在HEV病毒感染中的作用,对NCAM基因进行原核表达。选择原核表达载体pGEX-4T-1,因为其能在大肠埃希菌中表达融合蛋白,使重组蛋白具有生物活性。根据GenBank,设计NCAM基因引物,连接克隆载体pMD18-T Simple Vector,酶切鉴定后,连接表达载体pGEX-4T-1,诱导表达,根据标签蛋白进行亲和层析纯化。扩增NCAM基因片段为1281bp,诱导表达蛋白经Western blotting检测,具有生物活性。亲和层析纯化后的重组NCAM蛋白,经蛋白测定仪检测,蛋白纯度达92%,蛋白含量是15.43μg/ mL。重组NCAM蛋白多克隆抗体的制备及NCAM蛋白抗体抑制病毒增殖效果研究。免疫家兔制备多抗,将重组NCAM蛋白对家兔进行三次免疫,制备多克隆抗体。用重组NCAM蛋白包被ELISA板,检测家兔的抗体效价为1:16000;阴性对照未检出效价。用饱和硫酸铵方法纯化多抗,将制备的多抗加入神经细胞预先感作,然后接HEV。接毒36h后,试验组细胞培养上清液中的HEV血凝效价是1:20,对照组细胞培养上清液中的HEV血凝效价是1:23,两组间血凝效价相差三个梯度,差异显著。接毒48h后,试验组细胞培养上清液中的HEV血凝效价是1:22,对照组细胞培养上清液中的HEV血凝效价是1:24,两组间血凝效价相差两个梯度,两组间有明显差异。结果显示,NCAM蛋白抗体可显著抑制HEV增殖。NCAM蛋白在细胞及组织中的分布。用间接免疫荧光方法确定NCAM蛋白在细胞内的具体位置。免疫荧光结果显示,NCAM蛋白主要位于细胞膜,表明NCAM蛋白主要在细胞膜表达。免疫组化结果显示,NCAM蛋白在中枢神经系统包括大脑、小脑和脊髓中均有表达,与HEV在猪中枢神经系统的分布特征基本相同。鉴于以上研究,发现NCAM蛋白在HEV感染神经细胞过程中可能起着重要作用。更多还原

【Abstract】 Porcine hemagglutinating encephalomyelitis coronavirus (HEV) is a member of the Coronaviridae family, which causes porcine encephalomyelitis. HEV predominantly affects 1～3 week-old piglets, with clinical piglets vomiting, exhaustion and obvious neurological symptoms as the main feature. The mortality rate is up to 20～100%. Since 1958, when the disease broke out in the Canadian province of Ontario for the first time, many countries have reported about it. Serological test results proved that it is common for the pigs to be infected by HEV, and the disease may have spread worldwide. In August 2006, the disease broke out in part of the pig farms in Argentina, resulting to 1226 deaths, with the morbidity rate up to 52.6%. In China, an HEV infection has been reported occurring in a pig farm in Beijing as early as in 1985, followed with reports from Jilin, Liaoning, Shandong, Taiwan, etc. The large-scale epidemics of HEV occurred in Taiwan in 1994 had a fatality rate of almost 100%, resulting to serious economic losses. Serological survey conducted by foreign scholars revealed that HEV infection in pigs is very common, with a worldwide distribution, and it has been a serious threat on the industry of pig farming. However, it is unclear how the HEV is affected in the organism. Just because of the unclearness of the infection mechanism, no effective means have been promoted for the prevention and treatment of the disease. In view of the epidemic trend of the disease and the harm caused in pigs in China, an active study on the pathogenesis of the HEV is of great theoretical and practical significance.So far, the pathogenesis of HEV is unclear. In order to better control the disease, or rather eliminate it fundamentally and thoroughly, research of the pathogenesis is particularly important. As the coronavirus is qualified to identify the corresponding receptors of a specific host cell and then infect the cells of particular species selectively, HEV receptors may exist in the lesions of the nervous system, especially at the surface of neural cells. And it is these receptors mediated the invasion of the virus into cells. However, HEV cell receptors and the receptor-mediated infection mechanism are still unclear. To investigate the infection mechanism of the porcine hemagglutinating encephalomyelitis virus in the neural cell receptors and the receptor-mediated infection mechanism, mouse neural cells were primarily cultured first in this study, and a T7 phage display cDNA library from neural cells was constructed, the library was screened with the eukaryotically expressed S protein and HE protein respectively, and four candidate receptors were obtained through the screening. After sequencing, matching and bioinformatics analysis, the neural cell adhesion molecule (NCAM) was preliminarily deemed to be relative to the HEV infection, and identified. The polyclonal antibody of NCAM was prepared. Results showed that NCAM protein antibodies could inhibit the proliferation of HEV in the cell. The distribution of NCAM protein in the cells and the tissue was detected with immunofluorescence and immunohistochemistry to find that NCAM proteins were expressed in the central nervous system, including the brain, cerebellum and spinal cord, which is basically identical with the HEV distribution in the central nervous system of pigs. This provided evidences for the determination of its receptor protein. This study paved the way for the further study of the molecular pathogenesis of HEV.Primarily cultured mouse neural cell: Seven one-day old newborn mice were executed by broking the neck, and the entire brain tissue was peeled out with ophthalmic forceps, treated with 0.25% trypsin digestion, and placed in a incubator with 5% CO2 at 37℃for culture. The newly isolated neural cells were observed with inverted microscope, to find that there were many cells and they were round, among which a small amount had short axons. After the cells were cultured for 4-5d, the axons of most of cells were visible, which were migrating closer to each other and forming a network. After being cultured for 6-8d, the network among the cells was denser, and the outline was clearer, and more stereoscopic. After being cultured for 8-10d, the cells were reticulated. It was the growth peak at the 13d-20d, the growing follicles tended to be stable, and the cells became mature. Then with the reduction of the number of cells, cells of vacuolar degeneration began to appear. Study showed that primarily cultured mouse neural cells at 13d-20d could be used to establish phage library.Construction of the T7 phage display library of the mouse neural cells: The total RNA of the mouse neural cell was extracted with the Trizol; the mRNA was purified with the Poly (A) Quick mRNA Isolation Kit; and then the T7 phage display library of in-vitro cultured mouse neural cells was constructed, by using the Novagen’s OrientExpress Random Primer cDNA Synthesis Kit, EcoRⅠ/ HindⅢEnd Modification Kit, Mini Column Fractionation Kit, DNA Ligation Kit and the T7 Select 10-3b Cloning Kit respectively, and following the kit instructions for operation, and the titer of the unamplified library was 6.4×107 pfu / mL, and the library capacity was 1.52×107 recons. PCR identification on 100 randomly selected plaques was conducted, the library recombination rate was 94%; 86% of the insert segments were greater than 0.3kb, and the titer of the amplified library was 9×1010pfu/mL. The amplified library could be used to screen HEV receptors.Screening of the receptor gene of hemagglutinating encephalomyelitis virus: After the mouse neural cells grew into single-layer, the amplified library was screened for 5 times using the eukaryotic ally expressed S protein and HE protein, and at the fifth round of screening, the supernatant fluid was decked. A single plaque was selected, and the phage DNA was extracted. With the extracted phage DNA as the template, PCR amplification was conducted, and the size of the inserted segment for electrophoresis was detected. In each screening, 100 plaques were randomly selected for PCR, and the results showed that more than 80 inserted segments were of the same size, accounting for more than 80% of the total selected plaques. Screened phage insert segments were sent to the biotechnology company for sequencing. After sequencing, four different kinds of genes were were screened after BLAST analysis. However, only the neural cell adhesion molecule (NCAM) was neural cell-related protein gene; the other three kinds of genes were expressed everywhere in the body, and could be excluded from the HEV receptors, thus these three kinds of proteins were not analyzed. The NCAM nucleic acid sequences were analyzed with the DNAStar software, and the amino acid sequences were analyzed with InterPro Scan and ScanProsite.Analysis revealed that the NCAM was member of the immunoglobulin super family, as well as member of cell adhesion molecules (CAMs). NCAM played important roles in cell-cell adhesion and cell-extracellular matrix interactions in both mature and developing nervous system. During the development, they were involved in the cell migration, axon guidance, target recognition, and synapse formation; while in the mature nervous system, they maintain synaptic connections, cell-cell contacts, and neuron-glia interactions. NCAM underwent post-translational modification during the development, resulting to the abundant addition of PSA chains on its extracellular domain. HcoV-OC43, BCV and PHE-CoV recognized sialic acid-containing receptors similar to those of influenza C viruses. The role of neural cells in adhering molecules was closely related to the neurotropicity of HEV.Prokaryotic expression of NCAM genes: To further study the role of NCAM protein in the HEV infection, the prokaryotic expression of NCAM genes was carried out. The prokaryotic expression vector pGEX-4T-1 was selected, because it could express the fusion protein in E. coli and bring the recombinant protein with biological activity. According to GenBank, NCAM gene primers were designed to connect the cloning vector, the pMD18-T Simple Vector; after the connection of expression vector pGEX-4T-1 was identified with restrictive endonuclease, the protein was expressed after induction, and the label proteins were purified by affinity chromatography. The amplified NCAM gene fragment was 1281 bp, the proteins that were expressed after induction were detected to be biologically active with Western Blotting. After being purified by affinity chromatography, the recombinant NCAM proteins were detected with protein analyzer, with a protein purity of 92% and a protein content of 15.43μg / mL.The preparation of recombinant NCAM polyclonal antibodies and the study on the effect of inhibiting the viral proliferation of the NCAM antibody: The polyclonal antibody was prepared with immunized rabbit; the rabbits were immunized for three times with the recombinant NCAM protein to prepare polyclonal antibodies. The ELISA plate was coated with recombinant NCAM protein; the antibody titer of the rabbit was detected to be 1:16000; no titer was detected in the negative control. The polyclonal antibody was purified with saturated ammonium sulfate, and the prepared polyclonal antibody was added to the neural cells in advance to make sense, and then inoculated with the HEV. 36h after the inoculation, the hemagglutination titer of the HEV in the supernatant fluid of the cell culture was 1:20 test group, and the hemagglutination titer of HEV in the supernatant fluid of the cell culture was 1:23 in the control group; a difference of three gradient existed between hemagglutination titers of the two groups, thus the difference was significant. 48h after the inoculation, the hemagglutination titer of the HEV in the supernatant of the cell culture of was 1:22 in the test group, and the hemagglutination titer of the HEV in the supernatant fluid of the cell culture was 1:24 in the control group;a difference of two gradients existed between the hemagglutination titers of the two groups, thus the difference was significant. The results showed that NCAM antibody significantly inhibited the proliferation of HEV.NCAM protein distributes in the cells and tissue. The indirect immunofluorescence method was used to determine the specific location of the NCAM protein in the cell. Immunofluorescence results showed that, NCAM protein mainly located in the cell membrane, indicating that the NCAM protein was mainly expressed at the cell membrane. Immunohistochemistry results showed that the NCAM protein was expressed in the central nervous system, including the cerebrum, cerebellum and spinal cord, which is identical with the distribution of HEV in the central nervous system of the pig. Based on the above study, we found that NCAM protein may play an important role during the HEV infection on the neural cells or in the HEV proliferation.更多还原