| 摘要: |
| [目的]利用基因工程技术寻求能够高效、稳定表达猪α干扰素,且表达产物易于纯化的表达系统.[方法]以含猪α干扰素(IFN-α)基因的重组质粒pGEM-T-IFN-α为模板,PCR扩增猪α干扰素基因.将IFN-α片段定向插入原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-IFN-α,转化大肠杆菌BL21(DE3),在IPTG诱导下表达可溶性的融合蛋白(GST-IFN-α).[结果]SDS-PAGE可检测到相对分子质量约为45 ku的GST-IFN-α,Westernblot证实GST-IFN-α能与猪IFN-α单克隆抗体发生特异性反应.重组猪α干扰素经谷胱苷肽Sepharose-4B亲和柱层析纯化后,在Vero细胞上抗水泡性口炎病毒的活性为2.24×103U/mg.[结论]建立的表达系统能够表达重组猪α干扰素,表达的重组猪α干扰素具有一定的生物活性. |
| 关键词: 猪α干扰素 原核表达 抗病毒活性 |
| DOI： |
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| 基金项目:国家“十五”重大专项科技攻关计划项目（2002ba514a2-2） |
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| Expression of porcine interferon-α in Escherichia coli and detection for its antiviral activity |
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| Abstract: |
| 【Objective】 The study developed expression system which can express purified porcine interferon alpha steadily and easily.【Method】 Porcine interferon-alpha (PoIFN-α) gene was amplied taking recombination plasmid pGEM-T-IFN-α as template.PoIFN-α gene was inserted into pGEX-4T-1 of prokaryotic expression vector pGEX-4T-1 to form recombinant plasmid pGEX-IFN-α.pGEX-IFN-α was transformed into E.coli BL21(DE3)，which can express fused protein GST-IFN-α in E.coli BL21(DE3) by IPTG inducing.【Result】 The expressed product was identified by SDS-PAGE and Western blot．The resltuts showed that fusion protein GST-IFN-α was about 45 ku and it could react with monoclonal antibody for PoIFN-α.After purificating with Glutathione Sepharose-4B affinity column,the activity of recombinant PoIFN-α was detected by inhibiting the cytopathic effect.The result showed that the activity of recombinant PoIFN-α against vesicular stomatitis virus at vero cell was 2.24×103 U/mg.【Conclusion】 Expression system pGEX-IFN-α can express recombinant PoIFN-α，and recombinant PoIFN-α has bioactivity. |
| Key words: porcine interferon-alpha prokaryotic expression anitviral activity |
