关键词: 信号引导肽, 穿膜肽, 基因克隆

Abstract: 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 EcoRⅠ-BamHⅠsite of vector plasmid 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 EcoRⅠ. 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 BamHⅠ/EcoRⅠ. 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 constructed successfully.

Key words: neurotrohphin-4, antennapedia, gene clone