| Canine parvovirus (CPV) is non-enveloped single stranded negative DNA virus, is a major pathogen of dogs with acute hemorrhagic gastroenteritis and puppies myocarditis. The disease has caused a great threat to the dog raising. To study the interaction between pathogen and host, it is an effective means to analysis differences of genome expression profile by using DNA microarray. For exploring the interaction mechanism between CPV and host cell, this study chose the canine kidney cells (MDCK) as a research model and use the host cell gene expression changes of infection in vitro to predict the CPV infection in vivo, lay the foundation for the immune response of cells and the mechanism of anti-viral genes study.First, to understand the biological characteristics and bioinformatics of Canine parvovirus, and lay on the biology background of host cell infection. Virus was isolated, the virus step growth curve was draw and TCID50was detected. Then seven overlapping fragments were designed and amplified by PCR and DNA sequencing technology, after concatenation the full-length sequence of the isolates was got. This test obtained isolates CPV-LZ1the full-length sequence5053nt contains the two ends of the hairpin structure, and includes almost all bioinformatics laid a strain of molecular biology background. TCID50determination was10-3.428/0.1mL, and combined with the analysis of step growth curve characteristics, laid the biological background of the strains. Furthermore, the MDCK cell model of persistent infection with CPV was established and laid the foundation for follow-up test.Second, in order to learn the expression profile differences in the virus-infected cells, explore the pathogenesis of the immune response and their anti-viral mechanism, MDCK cell lines with persistent CPV infection was established and expression profiles differences was got by using Affymetrix Canine the Genome Array gene chip2.0, and the random five genes differentially expressed in persistent infection with CPV were verified by using the Real Time PCR, consistent with the results of microarray hybridization. By functional gene clustering and the differential expression gene function analyzing, preliminary understand the genome expression changes in cells infected with virus.In this experiment, expression profiles differences in MDCK cells sustained infection CPV was established, it showed that359genes with1.5-fold difference of expression (1.53%of total number of genes) were obtained, in which193genes were up-regulated (0.84%), and166down-regulated (0.69%).Five random altered expression genes verified by using real time PCR showed the results consistent with the microarray analysis.GO functional cluster analysis showed that some genome altered involved in immune response, the growth cycle regulation or signal transduction and protease activity, but some others of function were unknown. Part of the regulated expression of genes related to immune response, such as chemokine (CCL28), major histocompatibility complex (MHC), proteasome and colony-stimulating factor (CSF2); another part of the regulated expression of the gene and peptide chain endo-enzyme activity, such as cathepsin (CTSS), matrix metalloproteinase (MMP); in the expression downward genes most associated with cell proliferation regulation process, including cell proliferation, differentiation and apoptosis, such as metalloproteinase inhibitor (TIMP-1), prostaglandin E, cell apoptosis-promoting factor (BCL2L14). Learned by analyzing the preliminary restrictive role of the CPV to host cells, causing part of the cell proliferation regulatory genes were down regulated, and the positive response of cells to infection, part of the immune response genes, endopeptidase activity of genes, the increase expression, provided the experimental basis to explore the pathogenesis of the virus and the antiviral pathway. |
PDF Full Text Request