PML-RARα融合基因相关蛋白原核表达载体的构建、表达和纯化

摘要

Objective To construct,express,and purify the prokaryotic expression vector for PML-RARα fusion gene related proteins.Methods PML-RARα-pET32a(+) plasmid was used as a template and 1 200 bp sequences of PML and RARα were amplified by polymerase chain reaction and subcloned into expression vector pET32a(+) to construct recombinant plasmid;then the ligated products were transformed into DH5α.The positive clone was propagated and the recombinant plasmids were extracted for further identification and sequencing.The correct recombinant plasmids were named after PML-Flag-pET32a(+) and Flag-RARα-pET32a(+) respectively.E.coli BL21(DE3) were transformed with PML-Flag-pET32a(+) and Flag-RARα-pET32a(+),respectively,and induced with IPTG for protein expression.Fusion protein with an N-terminal Histag could be purified by Ni2+-resin affinity chromatography.Purified protein was identified by SDS-PAGE and Western blotting.Results The purpose gene was got by PCR amplification;it was confirmed by EcoRI/HindⅢdouble enzyme digestion and DNA sequencing that the recombinant plasmids PML-Flag-pET32a(+) and Flag-RARα-pET32a(+) were constructed successfully.PML and RARα proteins were expressed efficiently after the recombinant plasmids had been transformed and induced in E.coli BL21(DE3);SDS-PAGE and Western blotting demonstrated that the purified proteins were interest proteins.Conclusions Construction of recombinant plasmids and expression and purification of the PML and RARα proteins laid a solid foundation for antibody preparation.%目的 构建PML-RAR o[融合基因相关重组表达质粒,在大肠埃希菌中表达可溶性蛋白并进行纯化.方法 利用聚合酶链反应(Polymerase Chain Reaction,PCR)以PML-RAR α全长质粒为模板分别扩增出长度均为1 200 bp的PML和RAR o[序列,并将它们分别插入PET32a(+)质粒中,构建重组表达质粒,化学法转化大肠埃希菌DH5α进行克隆,菌落PCR筛选阳性转化子,双酶切和测序鉴定重组质粒的正确性.正确重组的表达质粒分别命名为PML-Flag-PET32a(+)和Flag-RARα-PET32a(+).将正确重组的表达质粒转化感受态大肠埃希菌BL21(DE3),经异丙基β-D-半乳糖苷(IPTG)诱导表达,利用表达蛋白的组氨酸“标签”(His-tag)进行Ni2+-树脂柱亲和层析纯化,SDS-PAGE和Western blotting鉴定纯化蛋白质.结果 PCR扩增获得目的基因,重组表达质粒经EcoRI/HindⅢ双酶切和测序鉴定证明构建正确.转化感受态BL21(DE3)并经诱导后得到高效表达,SDS-PAGE和Western blotting显示纯化蛋白为目的蛋白.结论 成功构建重组表达质粒,并经诱导表达后可纯化出目的蛋白,为进一步的抗体制备等实验奠定了基础.