人釉原蛋白编码区基因真核表达载体PsecTaq2A-AMG的构建

Construction of the Eukaryotic Expression Vector PsecTaq2A-AMG for Human Amelogenin

目的 构建人釉原蛋白 (AMG)编码区基因真核表达载体PsecTaq2A_AMG。方法 采用PCR技术体外扩增AMG完整分泌肽编码区。将扩增产物与PsecTaq2A分别用BamHI和XholI行双酶切 ,将获取的AMG目的基因片段连接到双酶切后的PsecTaq2A ,构建重组质粒PsecTaq2A_AMG ,并对重组质粒进行鉴定。结果 ①PCR扩增产物经 1 5 %琼脂糖凝胶电泳 ,可见大小约 5 19bp的特异性条带 ,与预期结果一致。②重组克隆PsecTaq2A_AMG酶谱分析与预期结果一致 ,序列测定结果与GenBank中的人釉原蛋白序列完全一致。结论 用此方法可成功构建AMG编码区基因真核表达载体PsecTaq2A_AMG。

Objective\ The purpose of this study was to construct a eukaryotic expression vector for human amelogenin (AMG).Methods\ PCR was performed to amplify the AMG encoding region. Amplified fragments for human AMG were recovered and inserted into eukaryotic expression vectors PsecTaq2A. The recombinant plasmid PsecTaq2A_AMG was constructed and their positive clones were identified.Results\ ①Amplified products were checked by electrophoresis and the results were satisfactory. ②The recombinant plasmid PsecTaq2A_AMG was analyzed by restriction endonuclease mapping and DNA sequencing. The results of sequencing were consistent with those from GenBank.Conclusion\ The recombinant plasmid PsecTaq2A_AMG was successfully constructed with properly inserted DNA sequence encoding mature amelogenin.\;