阿尔茨海默病pcDNA3.1/APP595/596质粒的构建研究

Construction of human Alzheimer's disease' APP-expressing Plasmid

目的构建人类阿尔茨海默病淀粉样前体蛋白(APP)真核表达质粒,为深入研究该病发病机制提供基础。方法从原始质粒克隆模板中,利用PCR方法扩增目的基因APP695cDNA,将目的基因和目的载体pcDNA3.1分别酶切;纯化酶切产物后定向连接,并将其转化细菌感受态细胞,对生长出的克隆行PCR鉴定,PCR鉴定为阳性的克隆送测序并行比对分析。结果应用DNAStar软件对阳性克隆测序结果与GenBank中APP基因序列比对,证实原序列中的错义突变G已被突变回C,第4位点碱基定点突变完成。结论成功构建了真核表达质粒pcDNA3.1/APP595/596,为建立阿尔茨海默病转基因细胞模型奠定基础。

【Objective】To construct Alzheimer＇s disease＇APP-expressing vector.【Methods】APP was PCRamplified,digested by HindIII and XbaI,and ligated into purified pcDNA3.1 vector.This recombinant plasmid was transfected into E.coli SH5α by means of heat shock.The recombinant plasmid was identified by PCR,and the positive cloning was identified by DNA sequence analysis.【Results】The positive cloning gene sequence was aligned to the complete gene sequence of APP by DNAStar software.The result confirmed that the accidental mutation（C→G） had been mutated back（G→C）.【Conclusion】The recombinant pcDNA3.1/APP595/596 plasmid was constructed successfully,which lays the foundation for further study on the pathogenesis of Alzheimer＇s disease.