凋亡素基因重组腺病毒的构建  

Construction of a recombinant adenovirus vector containing Apoptin gene

作　　者：陈健[1] 李大江[1] 王曙光[1] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所,中国人民解放军西南肝胆外科医院,重庆400038

出　　处：《消化外科》2006年第4期269-273,共5页Journal of Digestive Surgery

基　　金：重庆市科委资助项目(No.2002012)

摘　　要：目的构建携凋亡素基因的腺病毒载体,为进一步研究凋亡素基因功能及其作用机制建立基础。方法Pmel酶切线性化已经构建好的含凋亡素基因的穿梭质粒pAdTrack-CMV-VP3,然后电转化到BJ5183-AD-1细胞中进行同源重组。PacⅠ酶切出含凋亡素基因的腺病毒DNA,然后转染293细胞进行包装获得重组腺病毒,运用RT-PCR鉴定重组腺病毒。用氯化铯梯度离心纯化病毒。用物理和生物方法确定病毒的滴度。结果PacⅠ酶切证实同源重组成功。绿色荧光观察证实病毒包装成功并且具有感染力,RT-PCR证实了重组腺病毒具有凋亡素蛋白的编码。重组腺病毒的浓度为84.52×109VP/ml,滴度为6.561×109PFU/ml。结论成功构建含凋亡素基因的重组腺病毒,它的滴度足以满足体内外实验。Objective To construct a recombinant adenovirus carrying Apoptm gene so as to provide basis for further studying Apoptin gene functions and ascertaining corresponding mechanism. Methods The shuttle vector pAdTrack-CMV-VP3 containing Apoptin gene was linearized with Pmel and subsequently electrotransformed into BJS183-AD-1 cells for homologous recombination. The DNA containing Apoptin gene of the recombinant adenovirus was obtained by digesting with PacI and then transfected into 293 cells, where the recombinant adenovirus containing Apoptin gene was identified by RT-PCR and purified by cesium choride density centrifugation. Finally, the titre of the recombinant adenovirus was determined by biological and physical assays. Results Digestion with PacI proved successful homologous recombination. Green fluorescence showed successful recombinant adenovirus with infectiveness. RT-PCR confirmed that the recombinant adenovirus had coding of Apoptin gene proteins. The titre of the recombinant adenovirus was 84.52× 10^9 VP/ml and 6. 561 ×10^9 PFU/ml. Conclusion The recombinant adenovirus vector containing Apoptin gene is successfully constructed, with its titre sufficient for in vivo and in vitro experiment.

关 键 词：凋亡素基因 腺病毒载体 基因治疗 

分 类 号：Q78[生物学—分子生物学]