CAJ | 학술논문

运用PCR技术特异性地扩增了人IL-12p40亚基cDNA的编码区序列，得到940 bp的扩增片段，将其克隆于毕赤酵母（Pichia pastoris）表达载体pPICZαC的ClaⅠ，XbaⅠ位点，构建成重组表达质粒pPICZαC-p40.经LiCl2转化，Zeocin抗性筛选，得到含多拷贝p40基因的酵母工程菌Pichia pastoris X-33/p40，在φ=0.5%甲醇诱导下，p40基因得到了分泌型表达，培养上清的SDS-PAGE及Western-blot均显示表达产物相对分子质量约44 000，与预计大小相符，薄层凝胶扫描结果表明，分泌型表达蛋白占培养上清总蛋白量质量的47%.
운용PCR기술특이성지확증료인IL-12p40아기cDNA적편마구서렬，득도940 bp적확증편단，장기극륭우필적효모（Pichia pastoris）표체재체pPICZαC적ClaⅠ，XbaⅠ위점，구건성중조표체질립pPICZαC-p40.경LiCl2전화，Zeocin항성사선，득도함다고패p40기인적효모공정균Pichia pastoris X-33/p40，재φ=0.5%갑순유도하，p40기인득도료분비형표체，배양상청적SDS-PAGE급Western-blot균현시표체산물상대분자질량약44 000，여예계대소상부，박층응효소묘결과표명，분비형표체단백점배양상청총단백량질량적47%.
A 940 bp coding fragment of human IL-12 p40 gene was amplified via PCR technique and then cloned into Pichia pastoris expression vector pPICZαC. After transforming the linearized recombinant expression vector into the yeast strain X-33,we obtained the engineered yeast strain Pichia pastoris X-33/p40.The p40 gene was secretly expressed after induction with 0.5% methanol. Both the result of SDS-PAGE and Western-blotting proved a 44 000 protein band in the supernatant of medium. Gel scanning revealed the expression product accounted for 47% of the total supernatant protein.