定点突变重组ING4腺病毒基因转染系统的载体构建及鉴定  

Construction and Identification of Recombinant hING4 Recombinant Adeno virus Mediat ed Gene Transfer Syst em by Site-Directed Mutagenesis

作　　者：耿俊颖[1] 刘德生[1] 王红兵[1] 徐海洋[1] 

机构地区：[1]徐州医学院附属医院肿瘤科,江苏徐州221002

出　　处：《中国现代医生》2009年第32期18-20,共3页China Modern Doctor

摘　　要：目的重组构建hING4(人ING4)氨基酸序列。方法运用定点突变技术,在鼠ING4的基础上,设计两对突变引物P1、P2和P3、P4及全长ING4上下游引物P5、P6,通过四轮PCR,将mING4基因序列进行人源化改造,获得了编码hING4氨基酸的基因序列。将获得酶切目的基因片段,连接到转移载体pAdTrack-CMV上,形成重组转移载体pAdTrack-CMV-ING4。重组转移载体经PmeI酶切后与pAdEasy-1腺病毒载体在BJ5183大肠杆菌中同源重组,得到重组腺病毒载体pAdeasy-1-pAdTrack-CMV-ING4,经PacI酶切后脂质体转染QBI-293A包装细胞,获得重组腺病毒Ad-ING4。结果测序和RT-PCR结果表明hING4基因构建成功。结论hING4基因重组腺病毒载体构建成功。Objective To construct a recombinant a recombinant human inhibitor of growth 4 adenovirus vector. Methods Using site-specific mutagenesis technique,mING4 gene was changed into hING4 gene after fourth cycle PCR. hING4 cDNA was introduced into the shuttle plasmid pAdTrack-CMV,the recombinant shuttle plasmid（pAdTrack-CMV-ING4）and the backbone plasmid（pAdEasy-1 ） were tinealized with PmeI digestion and then under co-transformation in bacteria E.eoil BJ5183. Then,the newly recombinant plasmid pAdEasy-l-pAdTrack-CMV-ING4 was linearized with Pae I and was transferred into QBI-293A cells to form Ad-ING4. The control virus Ad-nul] with GFP was constructed in the same manner. The recombinant adenoviruses were amplified in QBI-293A cells,and viruses were purified from these cells to obtain viral stock. Results Sequencing and RT-PCR results proved that recombinant hING4 gene was constructed successfully. Conclusion The recombinant adenovirus hING4 vector was constructed successfully.

关 键 词：ING4基因 点突变 腺病毒 载体 基因治疗 

分 类 号：R394[医药卫生—医学遗传学] R512.62[医药卫生—基础医学]