促血管生成素-1腺病毒表达载体构建及鉴定

Construction and identification of adenovirus expression vector modified by Angiopoietin-1

目的构建表达促血管生成素-1(Ang-1)的腺病毒载体,为研究Ang-1防治慢性缺血性疾病提供前期基础。方法通过PCR从pMD18-Ang-1质粒中扩增Ang-1基因,酶切连接入载体pDC315-GFP并转化大肠杆菌感受态细胞,PCR鉴定测序分析;借助AdMax腺病毒包装系统实现重组,提取质粒DNA并转染HEK293细胞,观察GFP荧光并以Western Blot检测目的蛋白的表达,以终点稀释法测定病毒滴度。结果重组质粒pDC315-GFP-Ang-1经PCR鉴定阳性克隆,测序分析比对正确,重组腺病毒转染HEK293细胞后可观察到GFP-Ang-1融合蛋白,病毒滴度1×1011PFU/ml。结论成功构建了表达Ang-1基因的腺病毒载体。

Objective This study was designed to construct a adenovirus vector that expresses angiopoietin-1（Ang-1） gene with recombinant DNA technology,and lays the basis for the following study on Ang-1 prevention and cure of chronic ischmic diseases.Methods Ang-1 gene was amplified from plasmid pMD18-Ang-1 with polymerase chain reaction（PCR）.The fragment was inseted to shuttle plasmid pDC315-GFP by digesting with restrictive enzymes and linking reactions.The recombinant plasmid was transformed into E.coli DH5α,and identified with PCR and sequence analysis.The plasmid DNA extracted from AdMax auxiliary packaging system was co-transfect to HEK293 cells.The expression of GFP fluorescence and Ang-1 protein were detected by fluorescence microscope and Western blot.The titer of recombinant adenovirus was detected with end-point dilution method.Results The positive recombinant plasmid pDC315-GFP-Ang-1 was successfully screened by PCR,and the sequence of Ang-1 gene was further confirmed.The expression of GFP fluorescence and Ang-1 protein were found in HEK293 cells transfected with recombinant plasmid.The titer of recombinant adenovirus was about 1×1011PFU/ml.Conclusion The adenovirus vector expressing Ang-1 gene is successfully constructed.