Expression Of Recombinant Porcine Interferon-?6 And Recombinant Porcine Interferon-? And Activity Of Anti Foot-and-mouth Disease Virus

Foot-and-mouth disease(FMD)has seriously endangered the health and sustainable development of the swine industry in China.At present,there is no specific medicine to treat or prevent this disease.Therefore,it is significant to develop a safe,efficient and antiviral biological agent.Studies have shown that porcine interferon alpha6(PoINF-?6)and porcine interferon beta(PoINF-?)have good antiviral activity.In this study,two recombinant porcine interferon proteins were prepared in vitro by molecular cloning and genetic engineering,and their biological activities were verified.The anti-foot-and-mouth disease virus activity test in vitro was carried out to provide basic reference data for the detection and prevention of porcine foot-and-mouth disease.The main steps are as follows:(1)In order to obtain PoINF-?6 and PoINF-?genes,one pair of primers were designed,respectively.PoINF-?6 and PoINF-?genes were amplified from total RNA of porcine peripheral blood lymphocytes induced by concanavalin through reverse transcription polymerase chain reaction(RT-PCR).The amplified fragments were ligated into pcDNA3.1 expression vectors.After restriction enzyme digestion and sequence determination,it was confirmed that the cloned fragments were PoINF-?6 and PoINF-?genes.The recombinant plasmids pcDNA3.1-PoIFN-?6 and pcDNA3.1-PoIFN-?were successfully constructed.(2)The recombinant plasmids pcDNA3.1-PoIFN-?6 and pcDNA3.1-PoIFN-?were transfected into CHO-S cells.After 12 days of culture,the supernatant was collected by centrifugation.SDS-PAGE showed that there were single purpose bands at 19 k Da and 23 k Da.After the supernatant was filtered with 0.22?m filter membrane,the recombinant porcine interferon?6(rPoIFN-?6)and recombinant porcine interferon?(rPoIFN-?)proteins were purified according to the operating instructions of nickel affinity chromatography column.SDS-PAGE and Western-blotting analysis showed that the specific bands were about 19 k Da and23 k Da,which were consistent with the previous identification results,indicating that the two proteins have good immunogenicity.(3)To explore the antiviral activity of purified rPoIFN-?6 and rPoIFN-?proteins,the antiviral potency of rPoIFN-?6 and rPoIFN-?on porcine kidney cells(PK-15 cells)against blister stomatitis virus(VSV)was carried out,and the antiviral effect of rPoIFN-?6 and rPoIFN-?on foot-and-mouth disease virus was discussed.The results showed that the antiviral titers of rPoIFN-?6 and rPoIFN-?against VSV on PK-15cells were 1.7×10⁷U/mg and 4.5×10⁷U/mg,respectively,and they had no toxic effect on PK-15 cells.After diluted 10⁵times,rPoIFN-?6 can inhibit the cytopathic effect of foot-and-mouth disease virus(FMDV)type A strain 10⁴TCID₅₀and type O strain 10³TCID₅₀on PK-15 cells;After 10⁷times'dilution,rPoIFN-?can inhibit the cytopathic effect of FMDV type A strain 10⁴TCID₅₀and type O strain 10³TCID₅₀on PK-15cells.In addition,this study found that 4.7 ng/?L rPoIFN-?6 and 7 ng/?L rPoIFN-?could Fsignificantly induce the expression of multiple ISGs in PK-15 cells.To sum up,the research results suggest that rPoIFN-?6 and rPoIFN-?can effectively inhibit the proliferation of FMDV in vitro,which is expected to be applied to the prevention and treatment of FMDV in clinical production.