Cloning and expression of interferon-alpha/gamma from a domestic porcine breed and its effect on classical swine fever virus.
To further evaluate the clinical impact of recombinant PoIFN-alpha/gamma, PoIFN-alpha/gamma genes from a Chinese domestic big-white porcine breed were cloned using PCR, and expressed in a high-level prokaryotic system. The antiviral activities of rPoIFN-alpha/gamma on vesicular stomatitis virus (VSV), porcine reproductive and respiratory syndrome virus (PRRSV), and classical swine fever virus (CSFV) were investigated in different cell lines. The cloned PoIFN-alpha gene encodes a protein of 166 amino acids and has been named PoIFN-alphac. In a comparison of PoIFN-alphac with reported PoIFN-alphaI genes, eight amino acid substitutions at positions 43 (F to L), 78 (N to D), 86 (Y to C), 104 (A to V), 118 (R to L), 128 (T to P), 151 (S to V), and 156 (R to T) were observed, and resulted in no potential N-glycosylation site in the deduced PoIFN-alpha amino acid sequences. In contrast to PoIFN-alphac, one nucleotide substitution was found at position 462 (A to G), hence 0.1% synonymity is specific for the PoIFN-gamma gene. Both PoIFN-alphac and PoIFN-gamma genes were inserted into a prokaryotic vector pQE30, and expressed in E. coli M15 (pREP4) or SC11103 (pREP4) with the N-terminal six consecutive histidine residues, respectively. rPoIFN-alphac and rPoIFN-gamma proteins were detected by SDS-PAGE and Western blotting analysis at 20.7 and 18.0 kDa, respectively. In addition, the rPoIFN-alphac and rPoIFN-gamma protein were purified using Ni-NTA metal-affinity chromatography, and their anti-VSV, anti-PRRSV, and anti-CSFV activities were surveyed in homologous and heterologous cell lines. The results suggested that rPoIFN-alpha and rPoIFN-gamma could inhibit classical swine fever virus and other important viral pathogens in different cell lines.
Xia
Chun
C
The 211 Key Laboratory of Veterinary Molecular Biology, College of Veterinary Medicine, China Agricultural University, Beijing 100094, PR China. xiachun@cau.edu.cn
Dan
Wu
W
Wen-Xue
Wu
W
Jian-Qing
Wan
W
Li
Wang
W
Tian-Yao
Yang
Y
Qin
Wang
W
Yi-Bao
Ning
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000187:drug effects / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019316:Porcine respiratory and reproductive syndrome virus / Q000187:drug effects / Q000502:physiology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D013552:Swine; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects*
D016898:Interferon-alpha; D011994:Recombinant Proteins; D012313:RNA; D007371:Interferon-gamma