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Gene cloning, expression and purification of human FLT3 ligand
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 10
- DOI:
- 10.3321/j.issn:1000-2790.2002.10.003
- Key Word:
- 配体;克隆,分子;基因表达;蛋白纯化
Abstract： 目的克隆人FLT3配体(Flt3 Ligand, FL)基因,并在大肠杆菌中对FL进行表达、纯化. 方法分离人外周血单个核细胞,提取总RNA,采用RT-巢式PCR扩增人FLT3配体胞外段基因,以双脱氧终止法进行DNA序列分析;将测序正确的目的基因克隆入大肠杆菌融合表达载体pRSET-B;重组质粒pRSET-B-FL转化大肠杆菌BL21(DE3),用IPTG进行诱导后,收集细菌,菌体裂解后,利用常压离子交换色谱的方法对表达蛋白进行纯化,并进行SDS-PAGE及Western-blot检测. 结果从人外周血单个核细胞中克隆得到长462 bp的FL基因,测序正确;经EcoRⅠ和HindⅢ酶切鉴定证实,FL成功地克隆到表达载体pRSET-B;构建的表达载体pRSET-B-FL在大肠杆菌中表达出Mr 24 000的融合蛋白,经常压离子交换色谱化后的融合蛋白的纯度达到90%,该融合蛋白具有FL抗原特性. 结论成功地克隆并表达了FL基因,并对融合蛋白进行了初步纯化,为大规模获得FL创造了条件.
- [1]王慧,韩苇,颜真,等.pGEX-e23 （scFv） PE40融合基因的构建及其在大肠杆菌中的表达[J].2001,(7).
- [2]A W, Wognum,T P, Visser,K, Peters,etc.Stimulation of mouse bone marrow cells with kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating c[J].2000,11(15).
- [3]S D, Lyman,K, Stocking,B, Davison,etc.Structural analysis of human and murine flt3 ligand genomic loci.[J].1995,11(6).
- [4]S D, Lyman,L, James,L, Johnson,etc.Cloning of the human homologue of the murine flt3 ligand: a growth factor for early hematopoietic progenitor cells.[J].1994,83(10).
- [5]H E, Broxmeyer,L, Lu,S, Cooper,etc.Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells.[J].1995,23(10).
- [6]A, Wang,S E, Braun,G, Sonpavde,etc.Antileukemic activity of Flt3 ligand in murine leukemia.[J].2000,60(7).
- [7]C, Hannum,J, Culpepper,D, Campbell,etc.Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs.[J].1994,368(6472).
- [8]Bhat SD,Phan DH,Remmele RL Jr,etc.Minimization of recombinant human Flt3 ligand aggregation at the Tm plateau: a matter of thermal reversibility.[J].1999,38(16).
- [9]S, Fukuda,L M, Pelus.Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34(+) cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis.[J].2001,98(7).
- [10]L S, Rusten,S D, Lyman,O P, Veiby,etc.The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro.[J].1996,87(4).
- [11]D H, Lynch.Induction of dendritic cells (DC) by Flt3 Ligand (FL) promotes the generation of tumor-specific immune responses in vivo.[J].1998,18(1-2).
- [12]Fay J,Kraus E,Caron D,etc.Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo.[J].2000,165(1).
- [13]P, Bj?rck.Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice.[J].2001,98(13).
- [14]A, Gratwohl,L, John,H, Baldomero,etc.FLT-3 ligand provides hematopoietic protection from total body irradiation in rabbits.[J].1998,92(3).