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5.5×107U/mg，而标准品的生物学活性为2.2×107 U/mg。经过三步层析纯化得到电泳纯的rhIL-11蛋白质。结论成功获得IL-11人工基因和稳定分泌重组蛋白的基因工程菌株KM71-2424，该重组蛋白的生物学活性显著高于大肠杆菌表达的标准品，并获得较高纯度的重组蛋白。%Objective To express recombinant human interleukin-11 (rhIL-11 ) in methylotropic yeast Pichia pas toris. Methods By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZα-A-IL-11 was constructed and introduced into Pichiapastoris by linearized electroporation. The rhIL-11 pro tein was identified by ELISAand 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 rnreached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5×107 U/mg and 2.2×107 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-1l in yeast supematant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity.
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