野生型DNA聚合酶β重组绿色荧光蛋白表达载体的构建

Construction of the enhanced green fluorescent protein eukaryotic expression vector carrying wild type DNA polymerase β gene

目的:构建携带野生型DNA聚合酶β基因(wtDNApolβ)的重组真核绿色荧光蛋白表达载体pEGFP-C3-polβ。方法:运用PCR方法,从质粒pcDNA3.1-polβ中扩增出wtDNApolβ的开放阅读框序列,T-A克隆至pGEM-T载体,再定向克隆至pEGFP-C3,重组质粒pEGFP-C3-polβ用限制性内切酶酶切鉴定,通用鉴定引物PCR扩增鉴定,并进行DNA序列分析。结果与结论:多种鉴定方法证实了重组载体pEGFP-C3-polβ中的wtDNApolβ序列与GenBank中已知的序列相符。携带wtDNApolβ的重组增强型绿色荧光蛋白表达载体pEGFP-C3-polβ构建成功。

Aim: To construct a recombined enhanced green fluorescent protein vector carrying wild type DNA polymerase β gene(polβ). Methods: The fragment of wild type DNA polβ gene was amplified using PCR from plasmid pcDNA3.1-pol β to be a template and cloned to pGEM-T vector by T-A clone method and then inserted into eukaryotic expression vector pEGFP-C3. The recombined plasmid was identified using restricted enzyme analyzing, PCR technique and sequencing. Results and Conclusion: It is proved that the wild type fragment inserted into pEGFP-C3 is identical with the known fragment. The wild type pEGFP-C3-polβ is successfully constructed.