突变型DNA聚合酶β真核表达载体的构建及其表达

Construction of eukaryotic expressing vector of mutation polβ and its express

目的构建含食管癌突变型DNA聚合酶β基因(DNA polymeraseβ,polβ)的真核表达载体pcDNA4-C-polβ,使其在polβ基因敲除的EC9706细胞中表达。方法根据食管癌突变型polβ基因序列,设计并合成一对特异性引物(引物1、引物2),通过PCR扩增获得含BamHⅠ和ApaⅠ的基因序列,将其克隆入T载体,PCR及测序鉴定无误后,将其插入真核表达载体pcD-NA4-C,从而构建了含突变型polβ基因的真核表达载体pcDNA4-C-polβ。将重组质粒以脂质体包裹的方式转染入polβ基因敲除的EC 9706细胞,G418筛选获得稳定转染的细胞株。结果经过PCR筛选及DNA测序鉴定,证实真核表达载体pcDNA4-C-polβ构建成功。筛选后的EC 9706细胞经HislinkTM蛋白纯化试剂盒纯化,仅在39kD位置有一条带,证明蛋白表达及纯化成功。结论成功构建了食管癌突变型polβ基因的真核表达载体pcDNA4-C-polβ,并纯化出polβ蛋白,为进一步研究polβ的功能奠定坚实的基础。

Objective To construct mutant po1 βeukaryotic expression vector and to transfect it into EC9706 cell of po1 β gene targeting. Methods To design a primer pair （primer 1/ primer 2） for ampiifying pol β,the gene containing BamH I and Apa I was amplified by PCR, then was coloned into T-vector. Recombinant was identified by PCR and by DNA sequencing. After that,it was subcloned into pcDNA4-C plasmid. The eukaryotic expressing vector pcDNA4-C-po1 β was obtained. It was transfected into EC9706 cells of pol β gene targeting. The EC9706 cell lasting transfected with pcDNA4-C-pol β was obtained by G-418 screening. Results The eukaryotic expressing vector pcDNA4-C po1 β was obtained by PCR and sequencing, po1 β protion was obtained from EC9706 cells which were screened by HislinkTM protion purification system,the result was identified by SDS-PAGE. Conclusion Mutant po1 βeukaryotic expression vector pcDNA4-C-po1 βis successfully constructed and po1 βprotion is purified, which lays a solid foundation for further studying po1 βfunction.