突变型人APP基因与增强型绿色荧光蛋白基因融合载体的构建

Construction of the mutant of human amyloid precursor protein and enhanced fluorescence protein fusion gene

目的构建携带突变型人淀粉样前体蛋白基因(APP695)与增强型绿色荧光蛋白(EGFP)基因融合载体。方法在NCBI上查找APP695的序列,设计引物。以pCB6质粒携带的野生型APP695序列为模板,以突变体引物扩增突变型APP695。将突变型APP695与pIRES2-EGFP连接并测序证实。结果经过酶切鉴定、PCR和DNA测序等方法,证实构建的pIRES2-EGFP/APP695突变型质粒载体序列正确。结论成功构建APP695-EGFP融合基因载体,为研究AD发病的分子机制和治疗时的药物筛选奠定基础。

Objective To construct and express a recombinant vector bearing fusion gene of human amyloid precursor protein （APP）695 mutant gene and enhanced fluorescence protein （EGFP） fusion gene. Methods The primers were designed according to the sequences of APP695 gene found from NCBI. Then the mutant APP695 was amplifed by PCR with pCB6 carrying wild human APP695 and acting as a template, the gene of mutant APP695 was ligated to pIRES2-EGFP. The recombinant plasmid of plRES2-EGFP/APP695 mutant was verfied by checking sequences. Results The exact sequences of plRES2-EGFP/APP695 mutant vector were confirmed by digestion of restriction endonucleases, PCR and sequencing. Conclusions The pIRES2-EGFPAPP695 mutant fusion gene recombination has been constructed successfully, which lays the foundation for further research of AD pathogenesis and screening new drug targets on AD therapy.