摘要
通过克隆人FLT3配体 (rhFL)基因 ,建立其原核高效表达系统 ,为大规模获得rhFL产品 ,促进干细胞体外扩增移植等新技术在临床的应用创造条件。分离人外周血单个核细胞 ,提取总RNA ,采用RT 巢式PCR扩增可溶型FL基因 ,以双脱氧终止法进行DNA序列分析 ;将目的基因与 pProEXHT载体连接 ,重组体引入大肠杆菌并在IPTG诱导下表达 ;亲和层析纯化表达产物 ,观察其刺激CD34 + 细胞增殖的情况。从人外周血单核细胞中克隆得到长 481bp的rhFL基因 ,测序结果与已知人FL基因序列一致 ;rhFL的表达量约占菌体蛋白总量的 15 % ,经亲和纯化纯度达 90 %以上 ;rhFL +G CSF +EPO刺激CD34 + 细胞增殖约 40 0倍。获得了rhFL基因及其在大肠杆菌中的高效表达 ,初步建立了产物纯化途径 ,纯化后的rhFL具有较强的刺激造血干 /祖细胞增殖的能力。
Clone human FLT3 ligand gene,stablish a highly efficient expression system of rhFL in E.coli and a suitable purification method of expression products.A cDNA encoding soluble FL was cloned through RT\|nested PCR from the total RNA extracted from human peripheral blood mononuclear cells and identified by analyzing the nucleotide sequences,then introduced into pProEXHT plasmid to express a 6×His\|FL fusion protein in E.coli. The fusion protein expressed in inclusion body was isolated,solubilized and refolded,and then purified by chromatography on a Ni\|chelating affinity column.Its activity was detected by stimulating CD34 + cells to expanse.RhFL gene with a length of 481bp was isolated.The expression amount of rhFL reached to 15% of total bacterial proteins and the purity of rhFL was 90% after MCAC.RhFL+G\|CSF+EPO stimulated CD34 + cells to expanse up to 400 times.The purified rhFL had a powerful activity to stimulate hematopoietic stem cells to expanse in vitro .
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第6期708-712,共5页
Chinese Journal of Biotechnology
关键词
FLT3配体
原核表达
造血干细胞
表达
FLT3 ligand, prokaryotic expression,hematopoietic stem cells
