期刊论文

Chen, X.

中文

中山大学学报(自然科学版)(中英文)

0529-6579

2001

40

2

72-74+100

10.3321/j.issn:0529-6579.2001.02.020

广东省自然科学基金

本校为其他机构

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%.

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 p-40 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...