Cloning,Expression And Anti-PRRSV Infection Of Porcine ?1 Interferon Gene

Interferon-? is a new type ? interferon which was found by Sheppard et al in 2003,the members of the family of type ? interferon include IFN IFN-?1,IFN-?2,IFN-?3 and IFN-?4.In cells,due to differences in the gene structure and transcriptional regulatory factors of different IFN-?,IFN-?1 was expressed earlier than the other three IFN-?,so IFN-?1 played an important role in early inhibition of virus proliferatio.In the current research study,IFN-?1has a good effect on antiviral reaction.The current study found that IFN-?1 has a good antiviral effect.In continuous passage of human lung epithelial cell line?Calu-3?,increasing the expression of IFN-?1 strongly inhibits the proliferation of influenza A H1N1 virus.Harbin veterinary research institute,Chinese academy of agricultural sciences found a porcine type ? IFN-?1 to the pig epidemic diarrhea virus have a stronger inhibitory effect,because of its antiviral effect on intestinal mucosal epithelial cells is more accurate than the type ? interferon.It provides a new idea for further developing new antiviral drugs.As a local small pig species in Gui Zhou province,congjiang xiang pig has high research value in the medical field because its genes are highly homozygous and highly similar to the human genome.the blood from Jiangxiang pigs was collected from the Guizhou pig breeding farm and the isolated peripheral blood lymphocytes were used to extract total RNA.RT-PCR amplified the IFN-?1 gene from cong jiang xiang pig and cloned and analyzed its sequence.Then constructing the eukaryotic expression vector and evaluating the biological function of its expression product.Finally,the antiviral activity of the porcine reproductive and respiratory syndrome virus was studied.The main research contents of this paper include:1.Cloning and bioinformatics analysis of pig IFN-?1 geneTo clone and proceed the biological information analysis of Congjiang xiang pig IFN-?1gene.A pair of specific primers were designed to amplify the CDS region of Congjiang xiang pig IFN-?1 gene.The IFN-?1 gene was amplified from lymphocyte cells by RT-PCR and then analyzed by bioinformatics tools.The full-length CDS region of Congjiang xiang pig IFN-?1gene was 576 bp and encoded 191 amino acids.The molecular formula of IFN-?1 protein was C₉₄₉H₁₅₄₃N₂₇₇O₂₇₀S₇ with a relative molecular mass about 21.38 k Da and p I 9.42.Protein secondary structure prediction showed that Congjiang xiang pig IFN-?1 protein contained abundant secondary structure and were mainly alpha helix?64.90%?and random coil?25.65%?.Protein tertiary structure mainly contained alpha helix.The results of gene homology and phylogenetic tree revealed that Congjiang xiang pig IFN-?1 gene had the closest relationship with sus scrof.The cloning and bioinformatics analysis of Congjiang xiang pig IFN-?1 gene provided the theoretical foundation for further studying its functions.2.The-eukaryotic expression of pig interferon IFN-?1In order to obtain recombinant IFN-?1 from Jiangxiang pig and achieve its expression in CHO-K1 cells,according to the obtained jiangxi pig IFN-?1 gene sequence,The eukaryotic expression plasmid p EGFP-Po IFN-lambda 1 was constructed and transfected into CHO-K1 cells.After transfection,collecting cells 24 h,36h and 48 h respectively.The anti GFP labelling mouse monoclonal antibody was one resistance,and the anti mouse Ig G?H+L?antibody was two resistance.The Western blot detection showed that the Po IFN-?1 was expressed at different times after the transfection,but the expression of 36 h was the highest.Further,the antiviral activity of 48 h supernatant was detected by MDBK/VSV microcytopathic inhibition assay.The bioactivity of the supernatant was 2.1×103 U/m L.3.Pig interferon IFN-?1 inhibits PRRSV strainsIn order to verify the effect of PoIFN-?1 on the proliferation of PRRSV,the quantitative detection of MARC-145 virus by fluorescence quantitative PCR,Po IFN-?1 eukaryotic expression product pretreated MARC-145 cell 24 h with 50U/m L,100U/m L and 150U/m L and inoculated PRRSV strains respectively.Using Collecting cell suspension after 12 h,24h and 36 h.The electron microscope observed that the CPE phenomenon of the cells decreased significantly with the increase of the expression product dose,and detected the proliferation of the virus.The results showed that Po IFN-?1 could obviously inhibit the expression of m RNA and the titer of the virus proliferation,and it was dependent on the dose and time.