【摘要】 目的构建由人MDR1启动子调控CD∷upp融合基因表达的重组腺病毒,为对耐药肿瘤体内外靶向基因治疗奠定基础。方法自行设计一对含XhoⅠ和HindⅢ酶切位点的MDR1引物,从外周血中提取人类基因组DNA,通过PCR扩增MDR1启动子并插入PGL3-enhancer载体上,获得PGL3-MDR1质粒。从PORF-CD∷upp质粒中切下CD∷upp基因,插入PGL3-MDR1中MDR1启动子下游,并从中切下目的基因MDR1-CD∷upp,克隆到腺病毒穿梭质粒中,将带目的基因的穿梭质粒pAdTrack-MDR1-CD∷upp。与含有pAdeasy-1的BJ5183菌电转化,筛选提取>30 kb的阳性克隆,经线性化后转染293细胞,通过观察绿色荧光蛋白(GFP)的表达及PCR扩增出重组腺病毒中的目的基因MDR1、CD∷upp等方法加以鉴定。结果成功构建了含MDR1-CD∷upp靶向自杀基因的重组腺病毒载体Ad-MDR1-CD∷upp,病毒滴度为3.0×1010pfu/ml。结论该重组腺病毒的构建为下一步研究其对MDR1耐药细胞系的特异性杀伤作用和对耐药肿瘤模型的靶向基因治疗提供基础。更多还原

【Abstract】 Objective To construct a recombinant adenovirus vector containing human mdr1-CD∷upp gene which regulated by mdr1 promoter.Methods The promoter of the human mdr1 gene amplified from human genomic DNA by PCR was inserted into pGEM-T and PGL3-enhancer vector respectively.CD∷upp gene derived from the plasmid PORF-CD∷upp was inserted into the backward position of mdr1 promoter in the PGL3-enhancer vector.Subsequently,the obtained mdr1-CD∷upp gene was subcloned into an adenovirus shuttle plasmid pAdTrack.After being identified by endonuclease,PCR and sequencing,the recombinant shuttle plasmid pAdTrack-mdr1-CD∷upp was co-transformed into BJ5183 cells with the adenovirus backbone plasmid pAdeasy-1 to obtain the homologous recombination.The adenovirus was generated in 293 cells.Methods such as PCR and fluorescence microscopy were employed to identify the generated recombinant adenovirus.Results Recombinant mdr1-CD∷upp adenovirus were constructed successfully and the titer of virus was 3.0×10¹⁰ pfu/ml.Conclusion The recombinant adenovirus Ad-mdr1-CD∷upp might be an important base of the further research on its target gene therapy of multidrug resistance in cancer.更多还原