SS321
2011年5月4日 by admin
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【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 furtheSS321r 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 bSS321etween 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 SS321
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