真核表达质粒pBABE-hygro-PLAP-1的构建及鉴定

Construction and confirmation of a recombinant eukaryotic expression plasmid pBABE-hygro-PLAP-1

目的:构建并鉴定含有plap-1基因和已知FLAG序列的真核细胞表达载体pBABE-hygro-PLAP-1。方法:采用PCR扩增PLAP-1的目的片段,然后连接到已知FLAG序列的载体p3×FLAG-CMV-10上;再次通过PCR扩增出PLAP-1的目的片段及已知序列的FLAG,最后将其插入真核细胞表达载体pBABE-hygro的多克隆位点上。对该重组体进行酶切鉴定和测序验证。结果:通过对重组质粒进行酶切鉴定以及DNA序列测定分析,证明真核表达载体pBABE-hygro-PLAP-1构建成功。结论:成功构建plap-1基因的真核表达载体,为进一步研究plap-1基因的功能奠定了实验基础。

PURPOSE：To construct and confirm a recombinant eukaryotic expression plasmid pBABE-hygro-PLAP-1 containing periodontal ligament-associated protein（PLAP）-1 and a known FLAG sequence.METHODS：Firstly,the targeted DNA fragment was amplified from pCAG_PLAP-1 by PCR,then PCR product was inserted into p3×FLAG-CMV-10 vector with a known sequence of FLAG.Secondly,plap-1 gene and FLAG were obtained by PCR amplification from p3×FLAG-PLAP-1,then PCR product was inserted to proper sites of vector pBABE-hygro.Finally,the recombinant plasmid pBABE-hygro-PLAP-1 was confirmed by agarose gel electrophoresis and DNA sequence analysis.RESULTS：The construction of recombinant eukaryotic expression plasmid pBABE-hygro-PLAP-1 was confirmed through restriction enzyme maping analysis and DNA sequencing.CONCLUSIONS：The recombinant plasmid of pBABE-hygro-PLAP-1 is successfully constructed.It can be applied to plap-1 gene function research in the future.