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猪IFN-γ基因的融合表达及其表达产物的生物学特性
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窦永喜，翟军军，李健，潘晓梅，景志忠，才学鹏*
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（中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室 甘肃省动物
寄生虫病重点实验室，甘肃 兰州730046）
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摘要：
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依照猪IFN-γ（pIFN-γ）成熟蛋白基因序列设计引物，将已扩增的pIFN-γ基因克隆至原核表达载体pGEX-4T-1中，构建了GST-pIFN-γ融合表达载体，该载体转化宿主菌BL21后用IPTG进行诱导表达并对诱导表达条件进行了筛选和优化，筛选出最佳诱导表达条件后大量诱导表达，可溶性的表达产物经GST琼脂糖凝胶亲和纯化；包涵体经DOC洗涤、SKL变性溶解、透析复性进行纯化。经SDS-PAGE和Western-blot分析，证实得到了高纯度的融合蛋白，该蛋白的分子质量为42ku。选择FMDV和PRV两种不同的病毒（RNA病毒和DNA病毒）进行GST-pIFN-γ抗病毒活性测定。结果表明,GST-pIFN-γ融合蛋白可有效抑制这两种病毒引起的细胞病变，其对FMDV的抑制作用远远大于对PRV的抑制。对GST-pIFN-γ融合蛋白的稳定性及机体毒副作用的研究表明，该pIFN-γ的稳定性有较大提高，而毒副作用大大降低。 |
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关键词：
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猪；IFN-γ基因；融合表达；蛋白纯化；生物学活性 |
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中图分类号：S 852.42：Q 786 文献标识码：
A 文章编号：1673-4696(2008)06-0525-06 |
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Fusion expression of porcine interferon-gamma protein gene and
bioactivities of its expressed protein |
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DOU Yong-xi,ZHAI Jun-jun,LI Jian,PAN Xiao-mei,JING Zhi-zhong,CAI Xue-peng
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(Key Laboratory of Animal Parasitology of Gansu Province/State Key Laboratory of Veterinary Etiological
Biology/Lanzhou Veterinary Research Institute，Chinese Academy of Agricultural Sciences,Lanzhou 730046,China) |
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Abstract: A pair of primers were designed according to the sequence of porcine IFN-γ mature protein gene.Then the amplified gene was cloned into pGEX-4T-1 vector and the fusion expression vector GST-pIFN-γ was constructed.The recombinant vector was transformed into Escherichia coli strain BL21.The recombinant strain was induced by IPTG and the induction conditions were optimized.Under the optimized conditions,the fusion protein GST-pIFN-γ was expressed.The soluble product was purified by GST aga-rose gel.The inclusion bodies were washed by DOC,and dissolved in SKL and subsequently renatured by dislysis.The purified recombinant protein was analyzed by SDS-PAGE and Western-blotting.The results showed that the purified fusion protein GST-pIFN-γ was obtained，and the recombinant protein was approximately 42ku in molecular mass.The antiviral activity of the protein was determined by inhibiting the cytopathic effect,and the result showed that the fusion protein could suppress the cytopathic effect caused by FMDV(RNA virus) and PRV（DNA virus）,and the anti-FMDV activity was higher than that of anti-PRV.In addition,the GST-pIFN-γ had the better stability and lower side effect.
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Key words: swine;IFN-γ gene;fusion expression;protein purification;bioactivity |
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