摘要
为了提高人重组白细胞介素 2的稳定性和活性以及减少毒副作用 ,有必要定向改造rhIL 2的分子结构 .用PCR法从白细胞介素 2 (IL 2 )cDNA全序列中扩增成熟的肽基因片段 ,并利用定点突变技术将人重组白细胞介素 2第 12 5位游离的半胱氨酸编码序列突变为丙氨酸序列 .编码 18位亮氨酸的序列突变为蛋氨酸序列 ;编码 19位亮氨酸的序列突变为丝氨酸序列 .突变型人白细胞介素 2 (MvIL 2 )基因与表达载体pPIC9K重组 ,酶切线性化后用Invitrogen转化毕赤酵母试剂盒导入酵母细胞进行整合 ,经筛选得到一高表达白介素 2的克隆 .SDS PAGE显示 ,表达量约占总量的4 5 7% .经Western印迹验证 ,重组人白介素 2有免疫活性 ;与野生型IL 2相比 ,所获得的突变型IL 2纯品的比活性为 4 0× 10 7IU mg蛋白 ,比天然型IL 2高 4～
In order to improve the stability and bioactivity of interleukin 2 and decrease its toxic side effects, it is necessary to reconstruct rhIL 2 for obtaining a high level expression of human IL 2 in Pichia pastoris. Mature human Interleukin 2 gene was amplified from IL 2 cDNA by PCR, using the technique of site specific mutagenesis with a synthetic oligonucleotide primer, where the codon for cysteine 125 of human IL 2 was replaced with alanine, leucine 18 with methionine and leucine 19 with serine. The gene was ligated with yeast expression vector pPIC9K. The constructed plasmid, pPIC9K MvhIL 2, was linearized by Sal Ⅰ and transformed into Pichia pastoris KM71 by Pichia EasyCom TM Transformation Kit. Through selection of G418 resistant transformation and expressing clones, a high expression transformant was obtained. Pichia pastoris yeast cells integrating the plasmid pPIC9K MvhIL 2 produced a high level of human IL 2 about 45 7% of the total yeast body protein with immunocompetence by Western blotting. The specific activity of the purified MvhIL 2 was 4 0×10 7 IU/mg protein, which was 4～5 times of the NhIL 2.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2003年第5期618-624,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
重庆市科技委科研项目 (合同号:渝科委计 1999 16)~~
