摘要
目的:构建携带具有穿膜功能的融合报告基因NT4-GFP-Ant基因的重组腺相关病毒载体。方法:应用PCR技术和T载体克隆法克隆绿色荧光蛋白(GFP)基因;用T4DNA连接酶将测序正确的GFP与已构建成功的Ant基因片段及PBV220/NT4线性质粒载体定向连接,构建PBV220/NT4-GFP-Ant融合载体,酶切获取融合基因,并将其连入腺相关病毒载体PSSHG中,构建PSSHG/NT4-GFP-Ant重组腺相关载体并进行酶切鉴定。结果:T-easy/GFP经EcoRⅠ酶切后,可得到730bp左右的片段,GFP基因经DNA测序证实与GenBank序列一致;pBV220/NT4-GFP-Ant经BamHI/EcoRⅠ联合双酶切后,得到约为1000bp的基因片段;PSSHG/NT4-GFP-Ant经BamHI/EcoRⅠ联合双酶切后,得到大小约为1000bp长度的基因片段。结论:成功克隆GFP基因,成功构建NT4信号肽-GFP-Ant穿膜肽融合基因和PSSHG/NT4-GFP-Ant重组腺相关病毒载体。
Objective To construct NT4-GFP-Ant fusional reporting gene and the vector of NT4-GFP-Ant recombinant adeno-associated virus （AAV）. Methods The GFP gene was cloned by using PCR and T-vector cloning method. The positive clone was identified by the restriction enzymes, and then the cloned amplified fragments were sequenced and analyzed, The resulting gene of GFP and Ant, PBV220/NT4 were connected by DNA ligase, and thus PBV220/ NT4-GFP-Ant was constructed, then the NT4-GFP-Ant fragment was gained and identified by the restriction enzymes. The resulting gene of NT4-GFP-Ant fragment was inserted into the EcoR Ⅰ - BaraH Ⅰ site of vector plasraid pSSHG to construct the vector of NT4-GFP-Ant recombinant AAV. Results A 730 bp fragment of DNA was gained when T-easy/GFP was cut by the restriction enzyme EeoR Ⅰ. The cloned GFP gene was coincident with the sequence in GenBank. A 1 000 bp fragment of DNA was gained when pBV220/NT4-GFP- Ant was cut by the restriction enzymes BaraHⅠ/EcoR Ⅰ. A 1 000 bp fragment of DNA was gained when PSSHG/NT4-GFP-Ant was cut by the restriction enzymes BamHⅠ/EcoRⅠ. Conclusion GFP gene is cloned successfully, NT4-GFP-Ant gene and PSSHG/NT4-GFP-Ant recombinant AAV vector are construeted successfully.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2007年第5期798-802,共5页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金资助课题(30371567)
吉林省科技厅基金资助课题(2005045)
吉林大学研究生创新项目资助课题(703050)
关键词
绿色荧光蛋白
信号引导肽
穿膜肽
基因克隆
green fluorescent protein
neurotrohphin-4
antennapedia
gene clone
