| 人重组白细胞介素11在毕氏酵母系统中的表达及纯化 |
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| 作者姓名： | Zhu JK Xu ZX Huang WD Ming TS Xie W Xu Y Zhang XG |
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| 作者单位： | 苏州大学免疫教研室, |
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| 基金项目： | 国防科工委科学基金(Y5573162)和国际原子能机构(IAEA)基金(CPR/9/025)资助
Supported by the Grant from the Ministry of Military Industry of China(Y5573162) and the
Grant from IAEA Foundation (CPR/9/025) |
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| 摘 要： | 目的在甲醇营养型毕氏酵母蛋白质表达系统中高效表达人白细胞介素-11(rhIL-11)，便于进一步开发。方法以人工设计合成的rhIL-11基因，构建表达载体pPICZαA-IL-11，经线性化后电转化导入毕氏酵母菌株KM71，甲醇诱导表达，用ELISA和SDS-PAGE检测发酵上清中IL-11的抗原性和表达量，用IL-11依赖的B9-11细胞株分析其生物学活性，采用疏水层析、离子交换和凝胶过滤纯化发酵上清中的IL-11。结果序列分析表明，克隆载体中IL-11人工基因序列与设计相符；基因工程菌株KM71-2424在摇瓶培养上清中IL-11的表达量超过60mg/L，生物学活性测定显示其比活性为5.5×10
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| 关 键 词： | 重组人白细胞介素－11 毕氏酵母 表达 分离 纯化 |
| 修稿时间： | 2000年12月5日 |
Expression and purification of recombinant human interleukin-11 in Pichia pastoris |
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| Zhu JK,Xu ZX,Huang WD,Ming TS,Xie W,Xu Y,Zhang XG.Expression and purification of recombinant human interleukin-11 in Pichia pastoris[J].Acta Academiae Medicinae Sinicae,2001,23(2):127-131. |
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| Authors: | Zhu J K Xu Z X Huang W D Ming T S Xie W Xu Y Zhang X G |
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| Institution: | Department of Immunology, Suzhou University, Suzhou 215007, China. |
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| Abstract: | OBJECTIVE: To express recombinant human interleukin-11 (rhIL-11) in methylotropic yeast Pichia pastoris. METHODS: By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZ alpha-A-IL-11 was constructed and introduced into Pichia pastoris by linearized electroporation. The rhIL-11 protein was identified by ELISA and SDS-PAGE analysis. The bioactivity was analyzed by B9-11 cell line. A combination of liquid chromatography was developed to purify the rhIL-11 from ferment supernatant. RESULTS: The nucleotide sequence analysis indicated that the sequence of cloned artificial IL-11 gene accorded with that of designed; the secreted yield of rhIL-11 by yeast Pichia pastoris KM71-2424 in flask reached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5 x 10(7) U/mg and 2.2 x 10(7) U/mg respectively. The rhIL-11 was purified to electrophoretic purity by a combination of liquid chromatography. CONCLUSION: The human IL-11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71-2424). The biological activity of IL-11 in yeast supernatant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity. |
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| Keywords: | recombinant human interleukin 11(rhIL- 11) Pichia pastoris expression purification |
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