| 巨噬细胞抑制因子在毕赤酵母中的表达及鉴定 |
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| 引用本文： | 吴永和,王桂玉,吴尔若,马旭. 巨噬细胞抑制因子在毕赤酵母中的表达及鉴定[J]. 中国计划生育学杂志, 2005, 13(7): 409-413 |
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| 作者姓名： | 吴永和 王桂玉 吴尔若 马旭 |
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| 作者单位： | 1. 国家人口计生委科学技术研究所,北京,100081;中国协和医科大学
2. 国家人口计生委科学技术研究所,北京,100081 |
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| 基金项目： | “十五”国家重点科技攻关计划(2002BA709B10)基金项目 |
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| 摘 要： | 目的:在毕赤酵母表达系统中获得巨噬细胞抑制因子(MIC-1)高效分泌表达,并鉴定其免疫活性。方法:从BeWo细胞系中提取总RNA,通过反转录PCR获得目的片段,然后将基因亚克隆入pPIC9k表达载体,线性化后转化GS115酵母细胞,经G418抗性筛选获多拷贝重组子并诱导表达,PCR鉴定目的基因的整合。表达产物用Westernblot鉴定生物活性。结果:经过G418筛选到34株阳性克隆,MIC-1蛋白最高表达量为6.332mg/L。结论:成功构建了MIC-1表达载体,在酵母中获得多拷贝高效表达,并且形成正确的蛋白结构。
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| 关 键 词： | 巨噬细胞抑制因子(MIC-1) 毕赤酵母表达系统 pPIC9k质粒 |
| 修稿时间： | 2005-05-09 |
Recombinant and Identification Macrophage Inhibitory Cytokine 1 in Pichia Pastries |
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| Wu Yonghe,Wang Guiyu,Wu Erruo,et al.. Recombinant and Identification Macrophage Inhibitory Cytokine 1 in Pichia Pastries[J]. Chinese Journal of Family Planning, 2005, 13(7): 409-413 |
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| Authors: | Wu Yonghe Wang Guiyu Wu Erruo et al. |
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| Affiliation: | Wu Yonghe,Wang Guiyu,Wu Erruo,et al.National Research Institute for Family Planning,Beijing 100081. |
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| Abstract: | Objective: To obtain macrophage inhibitory cytokine 1 in pichia pastories, and to identify bioactivity of this protein. Methods: To Extract total RNA from BeWo cell line, and to attain gene of interest by RT-PCR, then to sub-clone this gene into the expression vector-pPIC9k, to transform into yeast GS115 after linearization, to screen multiple-copy transfomants by G418 and identify MIC-1 bioactivity by Western Blotting. Results: 34 strains of multicopy transforms were identified,the highest yield was 6.332mg/L.Conclusion: The protein is secreted in its corrected folded dimeric form at milligram per litre quanitities. |
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| Keywords: | Macrophage Inhibitory Cytokine 1(MIC-1) Pichia pastories expression system pPIC9k vector |
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