人重组粒细胞集落刺激因子突变体的构建、表达和其体内外生物学活性研究  

Studies on a mutant human granulocyte colony stimulating factor and its bioactivity both in vitro and in vivo

作　　者：李芳[1] 莫晓宁[1] 张颖妹[1] 袁岚[1] 饶严 范中玉 袁勇[1] 曹志敏 马大龙[1] 

机构地区：[1]北京医科大学卫生部医学免疫学重点实验室

出　　处：《中华微生物学和免疫学杂志》1999年第4期319-322,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家863计划生物技术领域基金

摘　　要：目的为提高人重组粒细胞集落刺激因子（ｈｕｍａｎｒｅｃｏｍｂｉｎａｎｔｇｒａｎｕｌｏｃｙｔｅｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇｆａｃｔｏｒ，ｒｈＧ－ＣＳＦ）的稳定性和活性，对ｒｈＧ－ＣＳＦ进行改造和体内、外活性研究。方法利用定向点突变技术，将人重组粒细胞集落刺激因子第１７位游离的半胱氨酸编码序列突变为丙氨酸序列，ＤＮＡ序列分析证明后，在大肠杆菌中表达突变蛋白。利用Ｇ－ＣＳＦ依赖细胞株ＮＦＳ－６０，对在不同温度及在人血浆中保存的Ｇ－ＣＳＦ蛋白（Ｇ－ＣＳＦ－Ａｌａ１７）和野生型的Ｇ－ＣＳＦ进行活性测定。腹腔注射后小鼠外周血白细胞计数检测其体内生物学活性。结果ＤＮＡ序列分析表明１７位半胱氨酸突变为丙氨酸，并在大肠杆菌中表达成功Ｇ－ＣＳＦ－Ａｌａ１７。纯化的突变蛋白对ＮＦＳ－６０细胞具有刺激活性；与野生型Ｇ－ＣＳＦ相比，在各种温度储存的突变蛋白保留更高的活性，与人血浆孵育５０小时后突变蛋白仍保持活性；小鼠一次性体内注射突变蛋白后外周血白细胞数明显高于野生型Ｇ－ＣＳＦ。结论Ｇ－ＣＳＦ突变体（Ｇ－ＣＳＦ－Ａｌａ１７）较野生型的Ｇ－ＣＳＦ具有更高的体外稳定性和体内造血刺激活性。Objective To reconstruct rhG CSF for improvement of its stability and bioactivity. Methods By using the technique of site specific mutagenesis with a synthetic oligonucleotide primer, we replaced the codon for cysteine 17 of human G CSF with alanine, confirmed by DNA sequencing and expressed the mutant protein (G CSF Ala 17) in Escherichia coli. The biological activity of the furified protein was determined with colorimetric microassay using G CSF dependent NFS 60 cells under different temperatures and in human plasma at 37℃. After intraperitoneal administration of the G CSF Ala 17 or wild G CSF, the peripheral leukocytes in normal mice were determined. Results The mutant G CSF was successfully constructed and expressed in E. coli. Purified G CSF Ala 17 protein had stimulating activity similar to the wild type of G CSF as assayed by G CSF dependent NSF 60 cells. However, the stability of the G CSF Ala 17 was superior than that of G CSF at different temperatures, and retained full biological activity after 50 hours culture in human plasma, as control. The G CSF Ala 17 has a higher stimulatory activity to the granulocytes than that G CSF after intraperitoneal administration into normal mice. Conclusions G CSF Ala 17 has an advantage over wild type of rhG CSF in storage stability and hemotopoietic activity in vivo.

关 键 词：定点突变 zhG-CSF 表达 生物学活性 

分 类 号：R392.12[医药卫生—免疫学]