人β地中海贫血IVSⅡ654(C→T)突变基因的克隆和真核表达体系的构建

Cloning of human β thalassemic mutation β IVS II654(C→T) and establishment of its mammalian expression system

目的 :克隆人 β地中海贫血IVSII654突变基因 ,构建该致病基因的体外真核表达体系。为该病基因治疗的研究提供理想模型。方法 :抽提 β 654纯合子患者DNA进行PCR扩增 ,将其克隆到pBGT51质粒中 ,然后将人 β珠蛋白基因座控制区 (LCR)及 β654基因亚克隆至真核稳定表达质粒pcDNA3 .1 +中。脂质体转染至小鼠红白血病细胞 (MEL) ,DMSO诱导MEL细胞表达 ,逆转录PCR检测人 β 654基因在MEL中的表达。 结果 :成功构建了几乎真实模拟人体内 β 654基因调控的真核表达系统。 结论 :β 654基因在MEL细胞中的表达情况同人体内 β654基因的表达完全一致 。

AIM: To clone human β-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human β IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human β 654 gene isolated from the β thalassemia patients homozygous for the β 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human β LCR and β 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3.1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human β 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human β thalassemic mutation βIVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human β 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human β thalassemic mutation βIVS II654(C→T).