反转录病毒载体介导的人β-珠蛋白基因低水平表达的机制初探  

The Possible Mechanisms Underlying Low Expression of Human β-globin Gene Cloned in a Retroviral Vector

作　　者：董文吉[1] 祖振响 刘德培[1] 郝德龙 郭志晨 梁植权[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院医学分子生物学国家重点实验室,北京100005

出　　处：《生物化学与生物物理学报》2002年第6期763-768,共6页

基　　金：国家高技术研究发展计划 (863计划 )资助项目 (No .863 -Z02-02 - 02 )~~

摘　　要：以人 β 珠蛋白基因簇基因座控制区DNaseI高敏位点 2和高敏位点 3组合的微小基因座控制区作为调控元件 ,以 374bp第二内含子部分缺失的 2 .0kbβ 珠蛋白基因作为结构基因的反转录病毒重组体 ,通过建立 ψ 2产病毒细胞系和瞬时转染双向性包装细胞系 ψ A两种方法获得重组病毒。感染鼠红白血病细胞后 ,Southern杂交鉴定前病毒整合完整性 ,用RNase保护实验检测人 β 珠蛋白基因的表达 ,通过PCR产物测序确定前病毒结构。结果表明 ,在瞬时转染 ψ A来源的重组病毒转导和质粒转染的鼠红白血病细胞中 ,人 β 珠蛋白基因的表达水平分别达到内源性鼠α 基因表达的 (5 2 .4± 11.2 ) % (n =12 )和 (73.8± 14 .3) % (n =12 ,未确定整合拷贝数 ) ;而在 ψ 2产病毒细胞系来源的病毒转导的细胞中 ,表达水平低于 3% ,前病毒序列分析发现其高敏位点 2中出现一个点突变 ,这可能是人β 珠蛋白基因呈低水平表达的一个原因。Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human β-globin expression cassette in gene therapy of β-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human β-globin gene. A recombinant retroviral vector was constructed, which harbored 2.0 kb β-globin gene with a 374 bp deletion in intervening sequence II coupled with a mini locus control region (miniLCR) composed of DNaseI hypersensitive sites (HS) 2 and 3 from human β-LCR. The recombinant retroviruses were generated from an established ψ-2 producer cell line, and by transient transfection of amphotropic packaging cell line ψ-A, respectively. The integrity of provirus in transduced MEL cells was determined using Southern blot, and the expression of transferred human β-globin gene was detected using RNase protection assay. The structure of provirus was further analyzed by sequence analysis of PCR products from genomic DNA of MEL individual clone as template. The results demonstrated that the average expression of human β-globin gene was (52.4±11.2)% (n=12) and (73.8±14.3)% (n=12, without copy-number determination), compared with that of endogenous murine α-globin gene, in MEL cells transduced with the recombinant retrovirus from transient transfection of ψ-A and MEL cells transfected with the construct, respectively. In MEL cells transduced with virus from ψ-2 producer cell line, however, the average expression was less than 3%. A point mutation was detected in HS2 of provirus in MEL cell clone with low expression of human β-globin gene. The possible mechanisms involved in low expression, including position effect, DNA methylation and RNA interference are discussed in addition to the point mutation.

关 键 词：反转录病毒载体 介导 人β-珠蛋白基因 低水平表达 机制 Β-地中海贫血 基因治疗 位置效应 RNA干扰 

分 类 号：R346[医药卫生—基础医学]