乳腺癌特异性转导小肽融合表达载体的构建以及目的蛋白的表达  

Construction of a prokaryotic expression vector for breast cancer transductive peptide and expression in E.coli BL21(DE3)pLysS

作　　者：高嫦娥[1] 洪敏[1] 刘为青[1] 何敏洁[1] 缪延栋[1] 王苗[1] 吕加令[1] 董坚[1] 

机构地区：[1]昆明医学院第一附属医院生物治疗中心,650031

出　　处：《中国医药生物技术》2010年第3期177-180,共4页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30860330)

摘　　要：目的构建乳腺癌特异性转导小肽PI-增强型绿色荧光蛋白融合蛋白的原核表达载体,并进行目的蛋白的表达、分离和纯化。方法合成PI的DNA序列,将其定向插入到p EGFPN2中,经双酶切获取PI-EGFP的核苷酸序列,再将其克隆入原核表达质粒pET-28a(+),构建出pET-28a(+)-PI-EGFP的原核表达载体;重组质粒转化BL21(DE3)pLysS感受态细胞,经IPTG诱导表达,利用His-tag对蛋白进行分离纯化,SDS-PAGE电泳和蛋白质印迹免疫分析(Western blot)鉴定纯化蛋白质。结果成功构建了pET-28a(+)-PI-EGFP的原核表达载体;其经诱导后,在大肠杆菌中高效表达,目的蛋白以可溶性形式存在;SDS-PAGE分析显示,在相对分子量约33kD的位置出现目的蛋白条带,与理论值相符;经His TALONTM Cartridge亲和层析纯化获得了高纯度的重组的PI-EGFP融合蛋白,Western blot鉴定结果显示获得了目的蛋白。结论成功构建出乳腺癌特异性转导小肽PI-增强型绿色荧光蛋白融合蛋白原核表达载体,并能够表达出PI-EGFP的融合蛋白,为进一步研究小肽的乳腺癌特异性转导功能奠定基础。Objective To construct a prokaryotic vector for breast cancer specific transductive peptide（PI） and enhanced green fluorescent protein（EGFP）,aiming to produce fusion protein PI-EGFP ,which was induced and expressed in E.coli. Methods First construct an eukaryotic expression vector p EGFPN2-PI,to obtain fusion fragment PI-EGFP,then insert it into the prokaryotic vector pET-28a（＋） to get pET-28a（＋）-PI-EGFP,which was transformed into E.coli BL21（DE3）pLysS.Expression of E.coli BL21（DE3）pLysS was induced by IPTG.Purify the protein with His TALONTM Cartridge Purification Kit.The specific protein expression product was detected by SDS-PAGE and western blot. Results After sequencing identification,pET-28a（＋）-PI-EGFP was constructed successfully.The fusion protein was expressed effectively in E.coli BL21（DE3）pLysS after transformed by the vector and induced by IPTG.Besides,it is soluble.The specific fusion protein had an apparent related molecular weight of about 33 kD as indicated by SDS-PAGE analysis,which in line with the theoretical value.Hight purity specific fusion protein was purified by His TALONTM Cartridge affinity chromatograph and was identified by SDS-PAGE and western blot. Conclusion The PI prokaryotic vector with pET-28a（＋）-PI-EGFP can effectively express PI-EGFP fusion protein ,laying a foundation for further study of the transduction function about PI.

关 键 词：乳腺肿瘤 重组融合蛋白质类 绿色荧光蛋白类 

分 类 号：R737.9[医药卫生—肿瘤]