人白细胞介素12基因在巴斯德毕赤酵母细胞中的表达(英文)

人白细胞介素 12 (hIL 12 )是人体内具有多种生物学活性的免疫调节因子 ,由p4 0和p35两个亚基经多对二硫键连接而成 .根据hIL 12的结构特点 ,采用LiCl二次转化将hIL 12的p4 0和p35两亚基基因导入巴斯德毕赤酵母X33细胞中 ,并经同源交换分别插入酵母基因组AOX1区域 ,构建成含hIL 12双亚基基因的酵母工程菌PichiapastorisX33 p4 0 p35 .经 0 .5 %甲醇诱导 ,p4 0和p35两亚基在同一酵母细胞中得到了表达 ,并组装成具有生物学活性的hIL 12p70分子

Expression of Human Interleukin-12 Gene in Pichia pastoris

Human interleukin-12(hIL-12) is an immunoregulatory cytokine with multiple biological functions in human body, which consists of two disulfide-linked subunits, p40 and p35. Based on this structural character, yeast strain Pichia pastoris X33/p40 was constructed by transforming the expression vector pPICZαC-p40 into Phichia pastoris X33 competent cells via LiCl transformation. Then the recombinant expression vector pPICZαC-p35 was used to transform the Pichia pastoris X33/p40 with LiCl, the yeast strain Pichia pastoris X33/p40-p35 containing the integrated hIL-12 p40 and p35 cDNA was obtained by high concentration Zeocin selection (1 000 mg/L). After induction with 0.5% methanol, the hIL-12p40 and p35 subunits were expressed secretely and assembled as hIL-12p70 in the same cell. PHA-activated PBMC proliferation assay demonstrated that the hIL-12 expressed in Pichia pastoris had the bioactivity to induce the proliferation of PHA-activated PBMC. A new pathway to express a protein with two or more subunits through Pichia expression system was developed.