CAJ | 학술논문

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p40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p40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Pichia pastoris X33/p40-p35.经0.5%甲醇诱导,p40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.
인백세포개소-12(hIL-12)시인체내구유다충생물학활성적면역조절인자,유p40화p35량개아기경다대이류건련접이성.근거hIL-12적결구특점,채용LiCl이차전화장hIL-12적p40화p35량아기기인도입파사덕필적효모X33세포중,병경동원교환분별삽입효모기인조AOX1구역,구건성함hIL-12쌍아기기인적효모공정균Pichia pastoris X33/p40-p35.경0.5%갑순유도,p40화p35량아기재동일효모세포중득도료표체,병조장성구유생물학활성적hIL-12p70분자.
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.