APP基因真核表达载体的构建及其在SH-SY5Y细胞中的稳定表达

Construction the eukaryotic expression vector of Amyloid beta-protein precursor and its stable expression in SH-SY5Y cell line

目的构建淀粉样前体蛋白(APP)基因真核表达载体,转染SH-SY5Y细胞,建立稳定转染细胞系。方法从质粒pcDNAs-APP中经PCR扩增出APP基因,利用DNA重组技术将其插入到真核表达载体pcDNA3.1(+)中,经酶切和测序鉴定后,脂质体转染法转染SH-SY5Y细胞,通过G418选择培养,建立稳定转染细胞系,通过印迹法(Western blot)检测APP的表达。结果 pcDNA3.1(+)-APP重组体经PCR扩增后片段大小约为2 300 bp,与预期相同。测序结果与目的序列相同,重组体载体构建成功。Western印迹检测到SH-SY5Y细胞中APP的表达。结论成功构建pcDNA3.1(+)-APP真核表达载体并稳定转染SH-SY5Y细胞,成功表达了目的基因,为进一步研究阿尔茨海默病分子学机制奠定了基础。

Objective To construct the eukaryotic expression vector of Amyloid beta-protein precursor（APP） and transfect into SH-SY5Y cell,to establish the stable cell line.Methods The APP gene was amplified by PCR from plasmid pcDNAs-APP and was cloned directly into eukaryotic expression vector pcDNA3.1（＋）,and was detected by endonuclease digestion and DNA sequencing.Furthemore,the recombinant plasmid was transfected into SH-SY5Y cell line and the stable transfections were screened and cultured by G418.Finally the stable cell lines were identified by Western blotting.Results The APP eukaryotic vector was successfully constructed and confirmed by endonuclease digestion（about 2 300 bp） and DNA sequencing.The expression of APP protein was successfully detected by Western blotting.Conclusions Eukaryotic expression vector and the stable cell line of recombinant pcDNA3.1（＋）APP are constructed,which may be a useful cell model to explore the mechanism of Alzheimer′s disease（AD）.