人脑神经生长因子基因的体外扩增

PCR Amplification of Nerve Growth Factor (β-NGF) Gene in Humau Brain

作者设计并合成一对特异性DNA引物,采取聚合酶链反应技术,分别从人脑cDNA文库和人脑基因组DNA中成功地制备了神经生长因子β亚基(β-NGF)基因片段,为该基因表达载体的构建奠定了基础。

Nerve growth factor, animportant neutrition factor for neuronalsurvival, growth, development and main-tenance of neuronal functions, consists ofthree kinds of subunit (α_2, β and γ_2), butonly the subunit β-NGF has the biologicalactivity. β-NGF coding sequence is lo-cated in a single exon of NGF gen.eIn ourstudy, a pair of specific primers(29 mers)has been designed and synthesizedwith ABI 318 A DNA synthesizer. Theupstream primer includes an initiationcodon and the 1st to 20th base of theexon. The downstream primer includesthe complementary sequence of 334 to 354base of the exon and a stop codon. Thefull-length DNA fragment of β-NGF genehas been successfully obtained from humanbrain cDNA library in λ ZAPⅡ/EcoR Ⅰand human brain genomic DNA respective-ly by using polymerase chain reaction.The reaction program consists of denatur-ing at 93℃ for 1 min, annealing at 55℃for 1 min, and extending at 72℃ for 2min. Both amplified products of 350 bpin length coincide with the exon codingsequence and are enough for the analysisof restriction mapping, DNA sequencingand construction of expression vector.