吉林白鹅α干扰素在大肠杆菌中的表达及抗病毒活性检测

Expression of Jilin white goose interferon-α in Escherichia coli and detection for its antiviral activity

用PCR方法扩增了吉林白鹅α干扰素(IFN-α)成熟肽编码序列,将IFN-α片段定向插入原核表达载体pGEX-6p-1中,构建重组质粒pGEX-IFN-α,将重组质粒转入大肠杆菌BL21(DE3)感受态细胞里,在IPTG诱导下表达可溶性的融合蛋白(GST-IFN-α)。SDS-PAGE、Western-blot检测结果表明,重组吉林白鹅IFN-α融合蛋白(rGoIFN-α)的分子量大小约为43ku,能与IFN-α抗血清发生特异性结合反应。重组吉林白鹅α干扰素经谷胱甘肽Sepharose-4B亲和柱层析纯化后,在鸭胚成纤维细胞上抗鹅细小病毒的活性为3.32×105 U/mg,本试验结果表明,该表达系统能够表达重组鹅α干扰素,且表达的重组鹅α干扰素具有一定的抗病毒活性。

The interferon αgene of JiLin White goose was amplified from genomic DNA,the segment coding mature peptide of the GoIFN-α gene was cloned into prokaryotic expression vector pGEX-6p-1 to construct recombinant plasmid pGEX-IFN-α.pGEX-IFN-αwas transformed into E.coli BL-21（DE3） for the expression of fused protein GST-IFN-α in E.coli BL-21（DE3） by IPTG induction.The expressed product was identified by SDS-PAGE and Western-blot.The results showed that fusion protein GST-IFN-αwas about 45ku and it could react with interferon antibodies.After purification with Glutathione Sepharose-4B affinity column,the activity of recombinant GoIFN-α was detected by inhibiting the cytopathic effect in fibroblast cells infected with Goose parvovirus.The result showed that the recombinant GoIFN-α has bicactivity and its activity against Goose parvovirus（GPV） at the duck embryo fibroblast cell was 3.32×105 U/mg.