摘要
将RT－PCR扩增的人粒细胞集落刺激因子（hG－CSF）结构基因与表达载体pJGW1重组，转化含有pGP1－2质粒的E.coliDH5α，进行温度诱导表达。经SDS－PAGE分析，rhG－CSF表达量占菌体总蛋白量的30％以上。将其复性，经CM－52FF离子交换层析纯化，纯化后的rhG－CSF（纯度＞98％）经SDS－PAGE测定分子量约为19kD；经胰酶裂解、反相HPLC分析肽谱具有8条特异肽段；等电聚焦测定PI值约为5．8；免疫印迹实验证实其与标准hG－CSF具有相同的抗原反应特异性；NH2-末端氨基酸分析与文献报道一致，采用小鼠白血病细胞株NFS－60测定活性为I×108IU／mg。
The gene which encode the human granulocyte colong-stimulating factor (G-CSF) was amplified by RT-PCR and inserted into expression vector pJGW1. The recombinant hG-CSP (rhG-CSF) was expressed in E. nd DH5 a that contained plasmid pJGW1-hG-CSF and pGP1-2. More than 30% of total protein of rhG-CSF was expressed and the molecular weight of expressed protein was approkimately 19 kD tested by SDS-PAGE. Western-blot revealed that 19 kD protein showed same specific antigenicity as standard hG-CSF. The rhG-CSF was isolated and purified up to 98%purity by inclusion body isolation, re folding and CM- Sepharose Fast Flow ion exchange chromatography. The IEF patterns and peptide map of rhG-CSF were the same as the stundard ones.Analysis of N-terminal amino acid showed the right sequences according to Genebank.
出处
《中国生化药物杂志》
CAS
CSCD
1998年第1期1-5,共5页
Chinese Journal of Biochemical Pharmaceutics
基金
广东省自然科学基金!970322
