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|標題:||A missense polymorphism in porcine interferon-gamma cDNA affects antiviral activity of the protein variant||作者:||Fan, Y.H.
|關鍵字:||porcine IFN-gamma;polymorphism;functional SNP;antiviral activity;nitric-oxide production;respiratory syndrome;gene polymorphisms;nucleotide-sequence;virus-replication;escherichia-coli;ifn;expression;cells;proliferation||Project:||Molecular Immunology||期刊/報告no：:||Molecular Immunology, Volume 44, Issue 13, Page(s) 3297-3304.||摘要:||
We determined the interferon-gamma (IFN-gamma) cDNA sequence from three porcine breeds, Duroc, Landrance/Duroc hybrid, and Landrance breeds. Five single nucleotide polymorphisms (SNPs) of porcine IFN-gamma (PoIFN-gamma) were identified, respectively, at positions 269 (A/G), 376 (M), 426 (T/C), and 465 (T/C) of the coding sequence in Landrance/Duroc hybrid, and at position 251 (A/G) in Landrance breed. Among them, A269G and A251G polymorphisms resulted in Q67R and K61R replacements in the mature protein. PoIFN-gamma cDNAs of Duroc breed (PoWN-gamma-W) and Landrance/Duroc hybrid (PoIFN-gamma-M), which, respectively, encoded Q67 and R67, were introduced into a prokaryotic expression vector pET32 to express recombinant PoIFN-gamma-W (rPoIFN-gamma-W) and rPoIFN-gamma-M protein variants in Escherichia coli. The identity of both protein variants was further confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We then compared bioactivities of these two recombinant proteins. Although both recombinant protein variants exhibited comparable activities in ant proliferation of PK-15 cells and in nitric oxide (NO) induction of porcine peripheral monocytes, antiviral activity of rPoIFN-gamma-W protein was significantly higher (P < 0.001) than that of rPoIFN-gamma-M protein in a plaque inhibition assay using pseudorabies virus (PRV). IC50 values of rPolFN-gamma-W and rPoIFN-gamma-M protein in anti-PRV assay were determined as 5.3 +/- 1.3 and 9.3 +/- 4.3 nM, respectively. In conclusion, we have identified five novel SNPs in PoIFN-gamma cDNA, including two missense polymorphisms that result in Q6-/R and K61R replacements. Our results further demonstrate that Q67R can markedly reduce antiviral activity of the PoIFN-gamma protein. This is the first report that shows the functional SNP in the coding region of IFN-gamma. In the future, it is imperative to determine whether Q67R replacement in IFN-gamma may have disease association. (c) 2007 Elsevier Ltd. All rights reserved.
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