新型rhNDPK-A工程菌的构建及表达产物纯化研究

Construct New Type of Engineering Bacteria to Express rhNDPK-A and Study on Purification of Expressed Product

目的 :为简化纯化过程 ,获得有临床研究价值的rhNDPK -A蛋白 ,构建新型rhNDPK -A基因的表达质粒 ,利用 6×His标签以Ni+ -NTA亲和层析柱纯化蛋白。方法 :将抑癌基因nm2 3-H1从质粒PBVNMH1中亚克隆于带有纯化标签的表达载体pQE4 0中。IPTG诱导表达目的蛋白。通过镍离子螯合层析柱一步纯化法纯化目的蛋白。结果 :pQE - 4 0中亚克隆的nm2 3-H1序列完全正确 ;目的蛋白在大肠杆菌M15中的表达量可达 4 9.6 % ;Ni+ -NTA亲和层析柱一步纯化后蛋白纯度为 93%。结论 :构建了带有 6×His纯化标签的新型rhNDPK -A基因表达质粒pQE -nm2 3H1,所构建质粒能高效表达目的蛋白 ,利用Ni+ -NTA亲和层析柱简便高效地纯化了表达产物。

Objective:To simplify the purification process and to get useful rhNDPK-A for clinical study,we construct new type of rhNDPK-A expression plasmid,make use of 6×His tag and purify rhNDPK-A by affinity chromatography column containing Ni^+-NTA.Methods:subclone nm23-H1 from plasmid PBVNMH1 to expression carrier plamid pQE40 which contain 6×His purification tag.Induce the expression of rhNDPK-A with IPTG.Purify the expression product by one step with Ni^+-NTA affinity chromatography column.Results:Construst expression plamid pQE-nm23H1 containing 6×His which can expression aim protein in high level,and get purified rhNDPK-A with Ni^+-NTA affinity chromatography column.