Stable Expression Of Porcine Interferon-alpha, Interferon-beta & Interferon-gamma Gene In NDV LaSota Strain And Study On Its Antiviral Activity

Interferon (IFN) is a kind of cytokine with antiviral activities, anti-tumor and regulatory immunity functions. Generally, it is difficult to purify natural IFN directly. Viral disease of porcine which has a large hazardous in porcine breeding, was a kind of important disease of porcine breeding. The manufacture and development of porcine interferon gene engineering production have extensive application prospects.In this study, The IFN genes, which encode porcine interferonαandγprotein, were cloned from total RNA which was derived from Con A-stimulated-cultured PBMCs of swine by reverse transcription polymerase chain reaction (RT-PCR). In addition, the porcine IFN-βspecific sequence which was obtained from GenBank was synthetized. The porcine IFN open reading frame was inserted into the locus between the phosphoprotein (P) and matrix protein (M) of full-length cDNA genomic cDNA of NDV LaSota vaccine strain for expression. The recombinant NDV which can express porcine interferon protein was rescued from genomic cDNA clone by the reverse genetics techniques. RNA was extracted from recombinant NDV and the porcine IFN gene could be amplified by RT-PCR. BHK-21 cells infected by recombinant NDV were detected by indirect immunofluorescence assay and it was found that PoIFN mainly anchored in cellular membrane. Western-blot assay confirmed that porcine IFN were expressed and the recombinant proteins were about 20kDa. The antiviral activity of PoIFN was tested by CPE50 method. The recombinant porcine IFN had the ability to induce an antiviral state against Vesicular stomatitis virus expressing green fluroncent protein (VSV-GFP) in PK-15 cells. The results showed that the PoIFN had high antiviral activity and the antiviral activity is related to the concentration of the PoIFN.