国际医药卫生导报2017，Vol.23Issue(13)：1998-2001,2025,5.DOI:10.3760/cma.j.issn.1007-1245.2017.13.004
PML-RARα融合基因相关蛋白原核表达载体的构建、表达和纯化
Construction, expression, and purification of prokaryotic expression vector for PML-RARα fusion gene related proteins
摘要
Abstract
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.关键词
PML-RARα融合基因/重组质粒/蛋白表达/蛋白纯化/急性早幼粒细胞白血病/抗体制备Key words
PML-RARα fusion gene/Recombinant plasmid/Protein expression/Protein purification/Acute promyelocytic leukemia (APL)/Antibody preparation引用本文复制引用
杜琴,蔡嘉镜,蒋春萍,徐磊,张国元,王东生..PML-RARα融合基因相关蛋白原核表达载体的构建、表达和纯化[J].国际医药卫生导报,2017,23(13):1998-2001,2025,5.基金项目
川北医学院校级科研发展计划项目(CBY15-A-ZD07) （CBY15-A-ZD07）
Project of Scientific Research Development Plan of Chuanbei Medical College (CBY15-A-ZD07) （CBY15-A-ZD07）
