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猪IL-18基因的原核表达及重组蛋白的纯化
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裴仉福1，陈瑞爱1，温纳相2，唐秀英1，唐满华1，朱文冠1，李琳1，程含波1
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(1.广东大华农动物保健品有限公司，广东 新兴527439；
2.广东温氏食品集团有限公司，广东 新兴527439)
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摘要：
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以pcDNA3.1-pIL-18为模板，采用PCR技术扩增到了猪白细胞介素18(IL-18)的成熟蛋白基因，通过KpnⅠ+SacⅠ双酶切及连接反应，构建了pET32c-pIL-18原核表达质粒。经过限制性内切酶分析、PCR鉴定及DNA序列测定证实，重组质粒中的基因片段连接正确。之后，重组质粒转化大肠杆菌BL21(DE3)，于37℃、1.0mmol/L IPTG条件下诱导表达。菌体裂解产物经SDS-PAGE分析，在分子质量约为33ku处出现了预期的目的蛋白。用8mol/L脲对表达产物变性，经Ni2+-NTA柱纯化，透析复性，得到了纯化的IL-18蛋白。Western-blot分析证实，纯化的重组IL-18蛋白具有反应活性。上述研究结果为重组IL-18的应用奠定了基础。 |
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关键词：
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猪白细胞介素-18基因；原核表达；蛋白纯化 |
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中图分类号：S 852.42：Q 785 文献标识码：
A 文章编号：1673-4696(2006)10-0842-05 |
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Prokaryotic expression of porcine interleukin-18 gene and purification
of the expressed IL-18 protein |
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PEI Zhang-fu1, CHEN Rui-ai1, WEN
Na-xiang2, TANG Xiu-ying1,
TANG Man-hua1,ZHU Wen-guan1,LI Lin1, CHENG
Han-bo1 |
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(1.Guangdong Dahuanong Animal Health Product Corporation Limited, Xinxing 527439,China;
2.Guangdong Wen’s Foodstuffs Group Corporation Limited, Xinxing 527439,China) |
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Abstract: The mature protein gene of porcine interleukin-18 was amplified from recombinant plasmid pcDNA3.1-pIL-18 by PCR. The prokaryotic expression plasmid pET32c-pIL-18 was constructed through KpnⅠ+ SacⅠ digestion and ligation, and the recombination was identified by enzyme digestion, PCR amplification and DNA sequencing. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and expression of the protein was induced with IPTG at 37℃ overnight. SDS-PAGE analysis showed that the recombinant plasmid was highly expressed in E.coli, and molecular weight of the expressed protein was approximately 33ku. After denaturation with 8 mol/L urea, the recombinant IL-18 protein which was purified with Ni2+-NTA His Bind Resin was refolded by dialysis against PBS and water. Western-blotting analysis confirmed that the purified protein had immunogenicity. These results provide the foundation for the practical application of the recombinant IL-18. |
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Key words:cattle; porcine interleukin-18 gene; prokaryotic expression; protein purification |
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