一种新型重组双人类干细胞因子在昆虫细胞中的表达。

Expression of a novel recombinant dual human stem cell factor in insect cells.

作者信息

Han Junhai, Yan Xiaohui, Zhu Jie, Zhi Xiaoyong, Zang Yuhui, Shen Beifen, Qin Junchuan

机构信息

School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China.

出版信息

Protein Expr Purif. 2003 Oct;31(2):311-7. doi: 10.1016/s1046-5928(03)00214-6.

DOI:10.1016/s1046-5928(03)00214-6

PMID:14550653

Abstract

Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter. The Sf9 cells infected with the recombinant virus expressed rdhSCF up to 6000 U/10(6) cell in flask and 8300 U/10(6) cell in spinner flask. The rdhSCF was purified by two-step chromatography. The molecular mass of rdhSCF was examined by western blotting and HPLC analysis. The specific activity of rdhSCF was up to 3.1x10(6) U/mg, about 8.7 times as high as that of monomer rhSCF from Escherichia coli.

摘要

干细胞因子（SCF）是一种造血细胞因子，可促进造血细胞的存活、增殖和分化。构建了一种双人类干细胞因子（dhSCF）cDNA，它由一个全长人类干细胞因子cDNA加上一个截短的hSCF cDNA（1-145个氨基酸）组成，二者通过一个编码肽（GGGGSGGGGSGG）的区域相连。将dhSCF基因克隆到杆状病毒转移载体pAcSecG2T中，置于多角体蛋白启动子的控制下。用重组病毒感染的Sf9细胞在摇瓶中表达rdhSCF的水平可达6000 U/10⁶个细胞，在转瓶中可达8300 U/10⁶个细胞。通过两步层析法纯化rdhSCF。通过蛋白质印迹法和高效液相色谱分析检测rdhSCF的分子量。rdhSCF的比活性高达3.1×10⁶ U/mg，约为来自大肠杆菌的单体rhSCF的8.7倍。