TAT-BACEsp-GFP三融合基因慢病毒载体的构建与鉴定

目的 构建穿膜肽基因和BACE底物肽融合基因与绿荧光融合基因慢病毒载体,为进一步对阿尔茨海默病进行基因治疗奠定基础.方法 利用非对称互补引物/模板法制备两端含有酶切位点的TAT-BACEsp融合基因的cDNA,使之克隆到带GFP慢病毒表达载体质粒FUGW中,经限制性酶切和测序鉴定重组载体.结果 限制性内切酶酶切和DNA测序分析证实获得的TAT-BACEsp产物与设计的完全吻合;TAT-BACEsp准确克隆入FUGW的多克隆位点.结论 成功构建了TAT-BACEsp与GFP 融合基因慢病毒载体,为进一步对阿尔茨海默病进行基因治疗提供了适合的稳定转染的载体.

Construction and identification of TAT-BACEsp-GFP triple fusion gene expressed from recombinant lentiviral vector

Objective To construct the TAT-BACEsp-GFP triple fusion protein expressed from recombinant lentiviral vector.Methods The target fragment TAT-BACEsp,which was amplified by PCR by means of asymmetrical primer/template,was recombined to the downstream of CMV promoter of the lentiviral vector FUGW.The construction obtained was sequenced to verify the integrity of the reading frame and the fidelity of the sequence.Results The evidence of restriction enzymes digestion and DNA sequence analysis showed that the insert was TAT-BACEsp cDNA,consistent with the fragment TAT-BACEsp fusion gene we designed.Conclusion The lentiviral vector expressing TAT-BACEsp-GFP triple recombinant fusion gene has been successfully constructed,and it may be a reliable recombinant vector on the road to therapeutics for Alzheimer's disease.