Expression Of Recombinant Porcine Interferon-α6 And Recombinant Porcine Interferon-β 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-α6）and porcine interferon beta（PoINF-β）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-α6 and PoINF-βgenes,one pair of primers were designed,respectively.PoINF-α6 and PoINF-βgenes 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-α6 and PoINF-βgenes.The recombinant plasmids pcDNA3.1-PoIFN-α6 and pcDNA3.1-PoIFN-βwere successfully constructed.（2）The recombinant plasmids pcDNA3.1-PoIFN-α6 and pcDNA3.1-PoIFN-βwere 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 0.22μm filter membrane,the recombinant porcine interferonα6（rPoIFN-α6）and recombinant porcine interferonβ（rPoIFN-β）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-α6 and rPoIFN-βproteins,the antiviral potency of rPoIFN-α6 and rPoIFN-βon porcine kidney cells（PK-15 cells）against blister stomatitis virus（VSV）was carried out,and the antiviral effect of rPoIFN-α6 and rPoIFN-βon foot-and-mouth disease virus was discussed.The results showed that the antiviral titers of rPoIFN-α6 and rPoIFN-βagainst 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-α6 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-βcan 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/μL rPoIFN-α6 and 7 ng/μL rPoIFN-βcould Fsignificantly induce the expression of multiple ISGs in PK-15 cells.To sum up,the research results suggest that rPoIFN-α6 and rPoIFN-βcan effectively inhibit the proliferation of FMDV in vitro,which is expected to be applied to the prevention and treatment of FMDV in clinical production.