人神经生长因子β亚基慢病毒载体的构建

背景：糖尿病神经源性膀胱的发病与神经生长因子缺乏有关,β亚基是构成神经生长因子(NGF)3种亚基中惟一具有生物活性的亚基,慢病毒载体是基因治疗的理想载体。 目的：构建过表达人神经生长因子β亚基(β-NGF)基因的慢病毒载体。 方法：通过目的基因的获得,载体质粒双酶切,载体质粒与目的基因的连接将人β-NGF基因克隆到慢病毒载体质粒pGC-FU中,构建重组慢病毒载体质粒pGC-FU-β-NGF。观察人β-NGF基因的克隆情况,并进行重组慢病毒载体质粒pGC-FU-β-NGF的PCR鉴定和测序。 结果与结论：构建的重组慢病毒载体质粒pGC-FU-β-NGF进行PCR实际获得的产物与预计PCR产物大小一致,即初步判断为构建成功的重组质粒。所获得的β-NGF基因经测序后与GenBank报道序列完全一致。说明pGC-FU-β-NGF中携带有正确的β-NGF基因。结果证实,实验成功构建了携带人β-NGF基因的重组慢病毒载体质粒。

Construction of lentiviral expression vector carrying human human beta nerve growth factor gene

BACKGROUND:Recent studies have shown that pathogenesis of diabetic neurogenic bladder is associated with lack of nerve growth factor. Nerve growth factor (NGF) composed of three subunits is one of the most important neurotrophic factors. Beta subunit (beta-NGF) is the only biologically active subunit. Lentiviral vector is an ideal vector for gene therapy. OBJECTIVE:To construct lentiviral expression vector carrying human beta-NGF gene. METHODS:By the way of acquisition of the target gene, double restriction digestion of vector plasmid with Age Ⅰ/EcoR Ⅰ, connecting the target gene with vector plasmid, Beta-NGF gene was subcloned into the lentiviral vector plasmid pGC-FU, to generate the lentiviral expression vector plasmid, pGC-FU-beta-NGF. The cloning of human beta-NGF gene, and sequencing of the recombinant plasmid pGC-FU- beta-NGF were observed in the study. RUSULTS AND CONCLUSION:For the recombinant lentiviral vector plasmid pGC-FU-β-NGF undergoing PCR, when actual PCR products share the same size with the expected, the recombinant plasmid is thought to have been successfully constructed. The result of sequencing showed the sequence of the cloned beta-NGF gene was consistent with that was reported in the GenBank. Plasmid pGC-FU- beta-NGF carried the correct beta-NGF gene.