Cloning,Expression And Expression Of Porcine IFN-?3 Antiviral Activity Analysis

Interferon(IFN)has a variety of biological activities such as enhancing the vitality of natural killer cells(NK cells),macrophages and T lymphocytes,as well as antiviral,antitumor,and immunomodulatory functions.It is widely used as a broad-spectrum antiviral agent.Interferon can not directly kill or inhibit viruses in the process of functioning,but it can make cells produce antiviral proteins by interacting with cell surface receptors,so as to achieve the effect of inhibiting viral replication.In this study,RT-PCR technology was used to amplify porcine IFN-X3 gene(pIFN-?3)from porcine lung macrophages(CRL2845),and cloned pIFN-?3 gene into eukaryotic expression vector pcDNA3.1 to construct a recombinant plasmid pcDNA3.1-pIFN-?3 was transfected into 293 cells using liposomes.After 48 h,the transient expression of pIFN-?3 was analyzed by Western blot,and the supernatant after transfection was applied to PK15,MDCK,and Vero cells to perform pIFN-?3 anti-recombinant vesicular stomatitis virus(VSVG)expression of green fluorescent protein.Activity analysis;further clone the pIFN-?3 gene into the baculovirus shuttle vector pFastBacl,and transform the recombinant shuttle plasmid pFB-pIFN-?3 into DH10(?)Bac E.coli competent cells by heat shock method;use blue and white spot screening technology to obtain the recombinant Baculosome rB-pIFN-?3;Transfect the recombinant bacmid rB-pIFN-?3 into adherent SF9 cells by cationic transfection method to rescue the recombinant baculovirus rBV-pIFN-?3.Recombinant baculovirus was expanded to the third generation,and the expression of pIFN-?3 protein was detected by Western blot;pIFN-?3 was prepared in large quantities using a serum-free expression system,and the cell culture supernatant was collected,using sucrose pad ultracentrifugation,sucrose density Purified by gradient centrifugation strategy,Western blot was used to identify the expression of pIFN-?3,the activity was measured,100U pIFN-?3 protein was applied to PK15,MDCK and Vero cells respectively,and pIFN-?3 was used in vitro against VSVG and porcine epidemic diarrhea virus(PEDV)Analysis of antiviral activity.The results showed that the pIFN-?3 gene was successfully amplified in this study and successfully expressed in 293 cells;transient expression can inhibit the proliferation of VSVG on PK15,MDCK,and Vero cells;the recombinant bacmid rB-pIFN-?3 was successfully constructed,and pIFN-?3 protein expressed by the Baculovirus expression systemcan specifically react with the His-tag antibody,indicating that the pIFN-?3 protein is successfully expressed and is reactive;the measurement result of interferon activity is 1.3 × 105 U/mL,and 100 U pIFN-?3 protein is taken acts on PK1 5,MDCK and Vero cells respectively.The results show that pIFN-?3 interferon protein can inhibit the proliferation of VSVG on PK15,MDCK and Vero cells in vitro,and can also inhibit the proliferation of PEDV on Vero cells in vitro.