β-淀粉样蛋白真核表达质粒的构建及鉴定

Construction and identification of recombinant plasmid pcDNA3-β amyloid

目的构建β-淀粉样蛋白真核表达质粒,为进一步开展老年性痴呆DNA疫苗的保护性研究打下基础。方法提取Tg2576转基因鼠基因组DNA,PCR扩增β-淀粉样蛋白(Aβ1-42)基因,用限制性内切酶KpnⅠ/XhoⅠ分别对扩增产物和真核表达质粒pcDNA3.1酶切,将目的基因定向克隆到pcDNA3.1载体上;对重组质粒进行双酶切初步鉴定后进行序列测定。结果特异扩增出Aβ1-42片段,大小为126bp,片段成功插入pcDNA3.1载体中。经双酶切及序列测定结果表明Aβ1-42目的基因正确重组入pcDNA3.1载体中。结论成功构建Aβ1-42真核表达质粒。

Objective To construct a mammalian expression plasmid pcDNA3.1-β amyloid （pcDNA3.1-Aβ1-42）, and to lay a foundation for further studying the protective immunity of pcDNA3.1-Aβ1-42 as an AD, sDNA vaccine. Methods Isolation the genome DNA from Tg2576 mice and amplified by PCR. The gene and the plasmid pcDAN3.1 were digested with Kpn I and Xho I respectively, and the gene was cloned into the pcDNA3.1 directionally. The recombinant vector pcDNA3.1-Aβ1-42 was characterized by restriction enzymes digestion and sequencing identification. Results The destination gene, 126 bp long, was amplified by PCR distinctively and inserted successfully into the plasmid pcDNA3.1. Enzymes digestion and sequencing identification showed that Aβ1-42 recombined with pcDNA3.1 exactly. Conclusion The mammalian expression vector pcDNA3.1-Aβ1-42 is successfully constructed