高效的红细胞特异性表达系统的建立和优化  

Establishment and optimization of a highly efficient erythroid expression system

作　　者：潘欣[1] 狄升蒙[1] 张胜男[1] 张艳磊[1] 易红飞[1] 赵晓杰[1] 黄进[1] 张鸿声[1] 田爱[2,3,4] 王熙才[2,3,4] 

机构地区：[1]同济大学附属上海市肺科医院临床转化中心,上海200433 [2]昆明医科大学第三附属医院 [3]云南省肿瘤医院 [4]云南省肿瘤研究所,云南昆明650118

出　　处：《转化医学杂志》2013年第1期4-10,共7页Translational Medicine Journal

基　　金：国家自然科学基金(30972633;81060177;81272325);上海市浦江人才计划(12PJ1433700)

摘　　要：目的探讨慢病毒载体中红细胞特异性基因调控元件的优化对小鼠红白血病(murine erythroleu-kemia,MEL)细胞向红系终端分化期间组织特异性表达重组蛋白的影响。方法改变红细胞特异性β-珠蛋白启动子编码长度、改变β-珠蛋白基因位点控制区DNaseⅠ高敏位点(hypersensitive site,HS)的组合、删除β-珠蛋白3'端增强子或β-珠蛋白内含子IVS2,由此构建一系列由不同缺失组合的基因调控元件控制目的基因(如人凝血因子Ⅸ)表达的慢病毒载体,包装成重组慢病毒,测定病毒滴度,并分别感染小鼠MEL细胞。用O6-苄基鸟嘌呤(O6-benzylguanine,BG)-卡莫司汀[1,3-bis(2-chloroethyl)-1-nitrosourea,BCNU]联合筛选以富集感染的阳性细胞,用酶联免疫吸附测定方法检测目的基因的表达,评估不同基因调控元件组合对重组慢病毒载体所携带的目的基因表达的影响。结果经限制性内切酶谱分析和序列测定,构建的慢病毒载体结构正确;三质粒系统瞬时转染人肾胚胎293T细胞包装的重组自灭活慢病毒可通过长时间高速离心有效浓缩;用50μmol/L BG-50μmol/L BCNU联合筛选可有效富集感染的阳性细胞;经环六亚甲基双乙酰胺(N,N'-hexamethyle-nebisacetamide,HMBA)诱导第8天的106个小鼠MEL细胞中表达的重组人凝血因子Ⅸ平均质量浓度达到正常水平的3.8%(191 ng/ml)。结论通过适当减少调控元件优化慢病毒载体不仅有助于提高携带的外源基因片段长度、增加载体制备的有效性、实现温和提高目的基因产物达到治疗水平需求量的目的,而且为开展以红细胞特异性表达载体介导的携带蛋白类药物的红细胞治疗以及非血液病基因治疗奠定临床前的研究基础。Objective To construct and optimize recombinant lentiviral vectors （LV） for recombinant gene expression driven by erythroid-specific gene regulatory elements, to investigate their expression capacity in routine erythroleukemia （MEL） cells induced by hexamethylene bisacetamide （HMBA） to terminal erythroid differentiation. Methods The length of erythroid-specific β-globin promoter gene was modified, DNase Ⅰ hypersensitive site （HS） of β-globin gene cluster locus control region was recombined, the 3＇-globin enhancer or β-globin intervening sequence IVS2 was deleted. The recombinant gene-human factor IX （hFⅨ） expression was driven by β-globin in LV vectors with different deletions and recombination of gene regulatory elements. 293T cells were co-transfected by transfer, packaging and pseudo-envelope plasmids. The titers of LV vectors were quantified by real-time PCR method. The MEL ceils transduced by these LV vectors were enriched by positive selection with various concentrations of 06-benzylguanine （ BG ） and 1,3-bis （ 2-chloroethyl ） -1 -nitro- sourea （BCNU）. The hFⅨ gene expression level was determined by enzyme-linked immunosorbent assay （ELISA） after BG-BCNU selection and HMBA induction. The contribution of these gene regulatory elements in epythroid-specific gene expression was evaluated. Results The structures of these LV vectors were confirmed by restriction digestion analysis,as well as DNA sequencing. The recombinant LV vectors obtained through tri-plasmids transient transfection of human embryonic kidney 293T cells were effectively concentrated by overnight high-speed centrifugation. The transduced cells were enriched effectively by BG-BCNU selection （50 μmol/L for both dugs）. High level of hFⅨ expression was achieved on 8th day after induction by HMBA in 10^6 MEL cells transduced by LV vectors with various gene regulatory elements,with average concentration of 191 ng/ml,a level equivalent of 3.8% of physiological level in human. Conclusion Optimizati

关 键 词：慢病毒载体 人凝血因子Ⅸ 红细胞特异性 基因调控 

分 类 号：Q78[生物学—分子生物学]