Cloning And Expression Of Porcine Interferon-alpha Gene And Studies On Anti-HCV Activity Of Recombinant Protein

Porcine interferon-alpha (PoIFN-a) is a homodimoric glycosylated cytokine that plays important roles on anti-infectious, antipreliferetive, and immonomodulatory agent. In order to develop recombinant PoIFN-a preparations preventing porcine viral infectious disease, and to further expound the research of cytokines in the field of molecular biology, mature PoIFN-a was cloned and expressed in E.coli expression system in the study.A pair of primers containing Sph I and Hind III restriction sites were designed, according to the PoIFN-a gene in DDBJ/GenBank. Then PoIFN-a gene was cloned from porcine genomic DNA by PCR. The PCR product was inserted in E.coli clone vector pGEM T-Easy, and sequenced. A new subtype PoIFN-a was obtained. The PoIFN-a was cut from pGEM T-Easy/PoIFN-a, inserted in E.coli expression vector pQE30. Being identified by PCR assay, digestion assay and sequenced, the recombinant plasmid pQE30/PoIFN-a for intracellular synthesis of mature PoIFNa in E.coli was constructed.The E.coli strain JM109 harbouring the plasmid pQE30/PoIFN-a was grown for expression. The cells were harvested and total E.coli proteins were detected by SDS-PAGE to select expressed strains. Western-blot analysis of the gel that showed a band of similar size was the major protein immunoreactive with the anti-6xHis MAb. The rPoIFN-ct antiviral activity was assayed by inhibition of the cytopathic effect(CPE) of Vesicular Stomatitis Virus(VSV) on Mardin Darby Bovine Kidney cells (MDBK) or other cell lines. The rPoIFN-a anti-HCV activity was assayed.The results showed that a new subtype PoIFN-a was cloned. The mature PoIFN-a coding sequence lacking signal peptids is SOlbp, and the predicted mature protein is 166 amino acids and a stop codon in length. The recombinant protein with 6xHis were expressed with molecular weight of 20.7KDa. The highest antiviral litre of rPoIFN-a was l l08IU/mI. The rPoIFN-a protected PK-15 cells against virulent Hog cholera virus (HCV) infection in vitro. In conclusion, the experiment lays a fundation for lots of production of the interferon-alpha.