Wilson病ATP7B基因重组腺病毒载体Ad-ATP7B的构建  

Construction of ATP7B gene recombinant adenovirus vector Ad-ATP7B in Wilson’s disease

作　　者：汤其强[1] 梁秀龄[1] 黄文林[2] 黄必军[2] 刘然义[2] 

机构地区：[1]中山大学第一附属医院神经内科,广州510080 [2]中山大学肿瘤医院实验研究部,广州510060

出　　处：《中华神经医学杂志》2005年第6期541-544,共4页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金(3040147;30370509);卫生部重大项目基金(2001321);教育部留学归国人员启动基金(2003406)

摘　　要：目的构建Wilson病基因ATP7B的重组腺病毒载体。方法分别以BamHⅠ+SalⅠ双酶切pcDNA3.0/ATP7B和pDC315,将ATP7BcDNA目的基因片段和线性化的pDC315连接,定向克隆构建pDC315/ATP7B,以PCR和酶切的方法鉴定。pDC315/ATP7B与腺病毒骨架共转染293细胞构建Ad-ATP7B,PCR进行鉴定。结果经PCR和酶切鉴定证实pDC315/ATP7B构建成功。pDC315/ATP7B与腺病毒骨架共转染293细胞后见明显的毒斑,说明二者在293细胞中同源重组并包装成功。经PCR证实重组腺病毒Ad-ATP7B构建已完成。结论本实验成功构建了ATP7B外源目的基因序列完全正确的重组腺病毒载体Ad-ATP7B,为下一步采用ATP7B基因重组腺病毒载体对Wilson病进行基因治疗打下了基础。Objective To construct recombinant adenovirus vector carrying ATP7B. Methods pcDNA3.0/ATP7B and pDC315 were digested individually by double restriction enzymes BamHⅠand SalⅠfor 3 h. The ligation reaction was carried out using ATP7BcDNA(6.6 kb) and pDC315(3.9 kb) fragments,and the full-fragment was transfected into competent DH5α. Positive clone is screened out and confirmed by PCR and identificated by restriction enzyme digestion. Then pDC315/ ATP7B and adenovirus vector were cotransfected into 293 cells. Recombination plaques were picked up and screened by PCR. Results ATP7B gene recombination adenovirus vector has been constructed successfully,named as Ad-ATP7B. Conclusion Construction of Ad-ATP7B established the foundation work for gene therapy of Wilson’s disease through recombinant adenovirus in the future.

关 键 词：基因重组腺病毒载体 ATP7B WILSON病基因 293细胞 pcDNA3 BamHⅠ PCR 基因片段 定向克隆 细胞构建 酶切鉴定 同源重组 基因序列 基因治疗 共转染 线性化 骨架 

分 类 号：R742.4[医药卫生—神经病学与精神病学]