title: The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α.
abstract: CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.
authors.affiliation: Key Laboratory of Animal Disease Diagnostic & Immunology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Li
authors.initials: L
authors.lastname: Huang
authors.forename: Rui-bing
authors.initials: RB
authors.lastname: Cao
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.forename: Jian-chao
authors.initials: JC
authors.lastname: Wei
authors.forename: Hassan
authors.initials: H
authors.lastname: Isahg
authors.forename: Li-jie
authors.initials: LJ
authors.lastname: Song
authors.forename: Wei-yong
authors.initials: WY
authors.lastname: Zuo
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Wei-wu
authors.initials: WW
authors.lastname: Wang
authors.forename: Xiang
authors.initials: X
authors.lastname: Mao
authors.forename: Pu-yan
authors.initials: PY
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002942:Circular Dichroism; D016384:Consensus Sequence; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D011558:Herpesvirus 1, Suid / Q000187:drug effects; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia; D010957:Plasmids / Q000235:genetics; D019316:Porcine respiratory and reproductive syndrome virus / Q000187:drug effects; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D034421:Sus scrofa; D015854:Up-Regulation / Q000187:drug effects; D018116:Vesiculovirus / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D011994:Recombinant Proteins