rhIL-13在毕氏酵母中的表达及生物学活性研究

Expression of Recombinant Human Interleukin-13 in Pichia Pastoris and Its Bioactivity in Vitro

目的 在甲醇营养型毕氏酵母蛋白质表达系统中高效表达重组人白介素 - 13(rhIL - 13) ,便于进一步研究开发。方法 根据酵母对密码子的偏爱性设计并人工合成了人IL - 13编码基因片段 ,经T4DNA连接酶、PCR技术连接形成IL - 13cDNA全长序列 ;将其插入 pPICZαA质粒中 ,构建成 pPICZαA -IL - 13表达载体 ;该载体经SacI线形化后以电转化法导入GS115酵母菌株中 ,经YPD平板Zeocine抗性筛选获得IL - 13表达阳性菌株 ;用WesternBlotting和ELISA检测发酵上清中IL - 13的抗原性和表达量 ,B9-11细胞株分析其生物学活性。结果 15 %SDS -PAGE显示重组人IL - 13分子量约 13kd ,Western -blotting证实为人IL - 13,ELISA测定在摇瓶培养上清表达量达 15 μg/ml,其生物学活性同标准品一样具有剂量依赖性的刺激B9-11细胞株增殖的作用。结论 成功获得IL - 13人工基因和稳定分泌蛋白的基因工程菌株 ,该重组蛋白的生物学活性达到标准品的要求。

Objective To express recombinant human interleukin-13(rhIL-13) in methylotropic yeast Pichia Pastoris . Methods An artificial gene with the optinal codon usage of Pichia Pastoris for IL-13 was designed and synthesized by T4DNA ligase and PCR. The expression vector pPICZαA-IL-13 was constracted and introduced into Pichia Pastoris GS115 by electroporation after linearized with SacI.Recombinants were selected by plating cells on YPD/Zeocine plates. The recombinant protein secreted by the yeast was identified by 15% SDS-PAGE, ELISA and Westren Blotting .The bioactivity was analyzed by B 9-11 cell line. Results Based on the result of the 15% SDS-PAGE and Westren Blotting assay,an about 13kd recommbinant protein expressed in the yeast supernatant was identified to be recombinant human IL-13.The secreted yeild of rhIL-13 in flask reached 15μg/ml.The biology activity of the IL-13 in yeast supernatant was the same as that of E. coli standard determined by stimulating the proliferation of B 9-11 . Conclusion The gene of recombinant human IL-13 can be obtained and its protein successfully expressed in the Pichia Pastoris . The biological activity of the IL-13 in yeast supertant is the same as that of E.coli standard.