人分化抑制因子3基因原核表达载体的构建及融合蛋白的表达  

Construction of prokaryotic expression vector of Id3 and expression of fusion protein in E. coli

作　　者：贾丽[1] 李晓军[1] 

机构地区：[1]南京军区南京总医院解放军临床检验医学研究所,江苏南京210002

出　　处：《医学研究生学报》2008年第10期1018-1020,1025,共4页Journal of Medical Postgraduates

基　　金：江苏省"六大人才高峰"基金课题(批准号:050203D)

摘　　要：目的:在大肠埃希菌中表达和纯化人分化抑制因子3(Id3),为进一步研究Id3的生物学活性打下基础。方法:PCR扩增人Id3基因编码区的DNA片段,将PCR产物克隆至pGEM-TEsay载体中并测序鉴定。将Id3 cDNA片段重组入原核表达质粒pET-32a(+),转化入大肠杆菌BL21(DE3)中,用异丙基β-D硫代半乳糖苷(IPTG)诱导融合蛋白的表达,表达产物经Western blot鉴定,并通过镍亲和层析柱纯化His-Tag标记的融合蛋白。结果:成功地构建了Id3的原核表达载体。Western blot分析证实,在大肠杆菌BL21(DE3)中表达了His-Tag融合蛋白,亲和层析法纯化后获相对分子质量为34 000的Id3融合蛋白。结论:重组质粒pET32a(+)-Id3能在大肠杆菌BL21(DE3)中表达,镍亲和层析柱可纯化获得融合蛋白。Objective： To express and purify the inhibitor of differentiation（Id3） in E.coli.Methods： The DNA fragment in the coding region of the human Id3 gene was amplified by PCR and cloned into the pGEM-T Easy vector for sequencing.The Id3 cDNA fragment was then subcloned into the prokaryotic expression vector（pET-32a）（＋） and transformed into E.coli BL 21（DE3）.The expression of the histidine-tagged（His-Tag） fusion protein was induced with isopropy-β-D-thiogalactoside（IPTG）,confirmed by Western blot and purified by the immobilized Ni^2＋ absorption chromatographic column.Results： The prokaryotic expression vector of Id3 was successfully constructed.Western blot confirmed the expression of the His-Tag fusion protein in E.coli BL 21（DE3） and that of the Id3 fusion protein with the relative molecular mass size of 33 000 after purified by the affinity chromatographic column. Conclusion： Recombinant Id3 can be expressed in E.coli BL 21（DE3） and the obtained fusion protein can be purified by the Ni-affinity chromatography column.

关 键 词：分化抑制因子3 原核表达 融合蛋白 

分 类 号：R730.23[医药卫生—肿瘤]