应用Gateway技术构建过表达β-catenin基因的慢病毒载体  

Construction of a lentivector containing over-expressing β-catenin gene by multisite Gateway technology

作　　者：吴倩[1] 魏亚明[2] 李瑜元[1] 曹燕雯[1] 陈其慧[1] 

机构地区：[1]广州医学院附属广州市第一人民医院消化科,广州市临床医学研究所,510180 [2]广州医学院附属广州市第一人民医院输血科,510180

出　　处：《国际生物医学工程杂志》2013年第4期207-211,I0002,共6页International Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目（81070385）;留学回国人员科研启动基金项目（教外司留[2012]940号）

摘　　要：目的应用Gateway技术构建靶向过表达β-catenin基因的慢病毒载体，验证该载体的功能。方法利用多位点Gateway克隆技术，通过BP重组反应，构建穿梭载体pDown—Ctnnb1；再通过LR重组反应，将pUp—EF1A、pDown—Ctnnb1、pTail-IRES／DsRed—Express2和母载体pLV．Des3d．P／puro4个质粒构建成表达载体pLV．EX3d．P／puro—EF1A〉Ctnnb1〉IRES／DsRed-Express2；再转化到感受态细胞stbl3，经PCR筛选菌落阳性克隆测序鉴定。将成功构建的慢病毒表达载体转染293T细胞，用实时荧光定量PCR法验证重组载体对β—catenin基因mRNA水平的影响。结果菌落PCR筛选和DNA测序鉴定显示入门克隆和表达载体插入片段序列正确，实时荧光定量PCR证实β-catenin基因mRNA水平显著上调（P〈0．01）。结论成功构建能显著上调β-catenin基因mRNA表达的慢病毒载体。Objective To construct a lentiviral vector over-expressing β-catenin gene by muhisite Gateway technology and confirm its effect. Methods By using multisite Gateway clone technique, the entry clone of pDown-Ctnnbl was constructed using BP recombination reaction. Then, LR recombination reaction was performed among pUp-EF1A,pDown-Ctnnbl ,pTail-IRES/DsRed-Express2 and pLV.Des3d.P/puro to generate an expression clone of pLV.EX3d.P/puro-EF1A〉Ctnnb1 〉IRES/DsRed-Express2. In each step, PCR and sequencing analysis were used to verify the constructions. When it was verified that plasmids were transfected into 293T cells, PT-PCR was performed to determine the mRNA level of β-catenin gene. Results Both PCR and sequencing analysis revealed that β-catenin over-expression gene was inserted into the target site and the insertion sequence was perfectly corrected. The RT-PCR results showed that the expression of β-catenin gene was significantly up- regulated. Conclusion The lenvivirus-mediate β-catenin over-expression gene was successfully constructed..

关 键 词：多位点Gateway技术 β-catenin 过表达 慢病毒载体 293T细胞 

分 类 号：R574.62[医药卫生—消化系统]