The Expression Of Canine Interferon-beta And Its Effect Against Canine Parvovirus Replication

Canine parvovirus disease,as one of the most serious infectious diseases endangering the health of dogs,has a huge threat to the survival of dogs.CPV is constantly changing,which brings great challenges to the prevention and treatment of this disease.Interferon?IFN?is a multifunctional cytokine widely expressed in vertebrates,and has a series of biological activities such as anti-virus,anti-proliferation,anti-tumor and immunoregulation.IFN-?is an important intermediate inducer and effector molecule in innate and adaptive immunity.Its unique role in antiviral mechanism is that IFN-?and its subtypes can not be replaced.Existing animal interferons are mainly prepared by prokaryotic expression,which require denaturation and renaturation to obtain active proteins.The complex production process greatly limits the application of interferon in veterinary clinic.The interferon expressed in eukaryotic cells is closer to the structure of natural interferon.Compared with dog IFN-?expressed in E.coli,the interferon expressed in CHO has better antiviral activity,immunogenicity and less side effects.In this study,dog blood was collected from the Animal Hospital of Guizhou University.Total DNA was extracted from isolated peripheral blood lymphocytes.The canine IFN-?gene was amplified by PCR,cloned and sequenced.The canine IFN-?eukaryotic expression plasmid was constructed and expressed in CHO-K1 cells.The antiviral activity of canine IFN-?was further studied.The main contents of this paper include:1.Cloning and bioinformatics analysis of canine IFN-?geneTo clone and proceed the biological information analysis of canine IFN-?gene.A pair of specific primers were designed to amplify the CDS region of canine IFN-?gene.The canine IFN-?gene was amplified from lymphocyte cells by PCR and then analyzed by bioinformatics tools.The full-length CDS region of canine IFN-?gene was 561 bp and encoded 186 amino acids.The molecular formula of IFN-?protein was C₁₀₁₄H₁₅₉₃N₂₆₃O₂₉₀S₉ with a relative molecular mass about 22.40 kDa and pI 5.98.Protein secondary structure prediction showed that canine IFN-?protein contained abundant secondary structure and were mainly alpha helix?76.88%?and random coil?19.35%?.Protein tertiary structure mainly contained alpha helix.The results of gene homology and phylogenetic tree revealed that canine IFN-?gene had the closest relationship with Nyctereutes procyonoides.2.Construction of a eukaryotic expression vector for canine IFN-?and its expression in CHO-K1 cellsTo construct the eukaryotic expression vector for canine interferon-??IFN-??and to explore the expression efficacy of it in CHO-K1 cells.The target gene was amplified and cloned into pEGPF-C1 vector to construct the eukaryotic expression vector pEGPF-C1-IFN-?,and the positive recombinant plasmids were confirmed by PCR identifications,double enzyme digestion analysis and sequencing.The recombinant eukaryotic expression vector was transfected into CHO-K1 cells by liposome.SDS-PAGE analysis of the cell lysate revealed a protein band of 22 ku,which was consistent with the expected molecular weight of canine IFN-?.Western-blot confirmed that the expression product was able to specifically combined with anti-GFP labeled mouse monoclonal antibody and has good reactivity.In this experiment,the recombinant eukaryotic expression plasmid pEGPF-C1-IFN-?of canine IFN-?was constructed,and expressed it in CHO-K1 cells successfully.3.Detection the effect of canine IFN-?on CPV replicationAfter subculturing F81 cells,four experimental groups were set up:positive group,negative control group,blank control group and control group.In the positive group,the CaIFN-?eukaryotic expression product 1 mL was added,and the F81 cells were pretreated for 5 hours and then inoculated with CPV.In the negative control group,the empty vector pEGFP-C1 eukaryotic expression product 1 mL was added,and the F81 cells were pretreated for 5 hours and then inoculated with CPV.The blank control group was inoculated with CPV directly after 5 hours of passage,while the control group was not inoculated.Cell suspension was collected at 12h,24h and 36h after inoculation,virus was collected after repeated freezing and thawing for 3 times,and DNA was extracted.Real-time fluorescence quantitative PCR analysis of CPV-VP2 gene was carried out to observe the changes of virus quantity in three groups at different time.The results showed that compared with the negative control group,the virus replication in the positive group was inhibited to a certain extent,most obvious at 24 hours,and the degree of cytopathy was slight.