天府肉鹅IFN-α成熟肽基因的原核表达及抗病毒活性

Prokaryotic Expression of Tianfu Goose IFN-α Mature Peptide and its Activity Against Virus

参照已克隆的天府肉鹅IFN-α完整ORF基因序列设计引物,克隆成熟肽基因,与载体pET32a连接,构建原核表达载体,并在表达菌Rosetta中表达,SDS-PAGE分析鉴定,通过Ni-IMAC亲和层析纯化和稀释透析复性后,采用微量细胞病变抑制法分析测定IFN的抗病毒活性。结果显示,IFN-α成熟肽基因全长486 bp,共编码161个氨基酸;成功构建了原核表达载体pET32a-mGoIFN-α并得到表达,SDS-PAGE和Western-blot分析表达蛋白的大小为38 ku;纯化复性后的表达蛋白抗水泡性口炎病毒(VSV)活性的比活力为5.52×103U/mg,表明重组蛋白具有良好的抗病毒活性。

A primer was designed reference to the complete ORF of Tianfu goose IFN-α,the mature peptide was cloned and connected into pET32a to construct the prokaryotic expression vector.Then the IFN-α gene was expressed in Rosetta and identified by SDS-PAGE.After the expressed protein was purified by Ni-IMAC affinity chromatography and dialyzed to renaturation,its antiviral activity was determinated by micro cytopathic effect inhibition.The results revealed that IFN-α mature peptide gene was 486 bp and encoded a polypeptide of 161 amino acid.The prokaryotic expression vector pET32a-mGoIFN-α was constructed successfully and the gene was expressed.SDS-PAGE and Western-blotting assays demonstrated the expressed protein was about 38 ku.After purifying and renaturating,the antiviral activity of IFN was evaluated by using vesicular stomatitis virus（VSV） and had an antiviral specific activity of 5.52×103 U/mg,which Showed that the recombinant protein had a good antivital activity.