携带人脱嘌呤/脱嘧啶核酸内切酶(APE1)基因重组慢病毒载体的构建及体外表达检测  

Construction and identification of lentivirus vector encoding APE1 gene

作　　者：张璐[1] 方宇[2] 曾昭书[3] 连亚军[1] 许予明[1] 

机构地区：[1]郑州大学第一附属医院神经内科 [2]郑州大学第一附属医院急诊科 [3]郑州大学基础医学院法医学教研室,郑州450052

出　　处：《重庆医科大学学报》2011年第9期1073-1076,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:31071100)

摘　　要：目的:构建携带人脱嘌呤/脱嘧啶核酸内切酶-1(Apurinic/apyrimidinic endonuclease-1,APE1)基因重组慢病毒载体内英文摘要中,并检测其体外表达目的基因的水平。方法:应用基因克隆技术将人APE1基因克隆入慢病毒载体LV中,经过PCR、酶切和测序鉴定后,与包装四质粒系统共转染293T细胞,制备并收集携带APE1基因的慢病毒,重组慢病毒感染体外培养大鼠神经元细胞,利用实时定量PCR、蛋白质免疫印迹方法检测APE1的表达情况,观察病毒转染效果。结果:PCR、酶切和测序鉴定,成功克隆了LV-APE1重组质粒。包装慢病毒,浓缩病毒悬液的滴度为1.0×109 TU/ml。慢病毒颗粒稳定转染大鼠神经元细胞,当感染复数为50时,感染效率100%,实时定量PCR、蛋白质免疫印迹方法可以检测到重组慢病毒感染大鼠神经元细胞后能高水平表达APE1。结论:成功构建了携带人APE1基因慢病毒载体,并可在体外高水平表达其所携带的目的基因,为将其进一步应用于神经系统退行性疾病的治疗研究奠定了方法学基础。Objective：To construct lentivirus vector encoding human apurinic/apyrimidinic endonuclease-1（APE1） gene and examine its ability to express the APE1 gene in vitro.Methods：The specific APE1 sequence was cloned into the plasmid of LV to construct the APE1 expression plasmid LV-APE1.The recombinant plasmids were identified by DNA sequencing and restriction digestion.Then the packaging cell lines（293T cell）were cotransfected with the LV-APE1 together with the plasmids pRsv-REV,pMDlg-PRRE,PMD2G and interfered by phosphate-calcium deposit method.The recombinant lentivirus vector was used to infect the Wistar rat neuron cell.The expression of APE1 was detected by real-time quantitative PCR and Western blot.Results：The results showed the recombinant lentivirus vector carrying APE1 gene was constructed successfully;the virus titers were above 1.0×109 TU/ml,and its infection efficiency in the Wistar rat neuron cell was 100%.The recombinant lentivirus vector could up-regulate the expression of the APE1 gene.Conclusion：The lentivirus vector carrying APE1 gene can enhance the expression of APE1 gene significantly,which lays the basis for its application in the treatment of neurodegenerative disease.

关 键 词：人APE1 慢病毒载体 基因治疗 

分 类 号：Q781[生物学—分子生物学]