hCGβ-(CTP)_4和hCGβ-(CTP)_4-(C3d-P28)_4在大肠杆菌中的表达及纯化  

Expression and Purification of the Fusion Proteins hCGβ-(CTP)_4 and hCGβ-(CTP)_4-(C3d-P28)_4 in E.coli

作　　者：石燕[1] 蒋椒[2] 沈卫英[2] 蒋雅红[2] 申庆祥[2] 

机构地区：[1]复旦大学上海医学院,上海200032 [2]上海市计划生育科学研究所,上海200032

出　　处：《生殖与避孕》2007年第4期241-246,279,共7页Reproduction and Contraception

基　　金：本课题为国家自然科学基金(No.30472087);上海市科技重大攻关项目(No.05DZ19314)

摘　　要：目的:在大肠杆菌BL21(DE3)中表达hCGβ-(CTP)_4、hCGβ-(CTP)_4-(C3d-P28)_4融合蛋白,并对蛋白进行分离纯化。方法:将pBSMR-(CTP)_4中的(CTP)_4基因片断克隆入表达载体pET-28a(+),得到表达质粒pET-28a(+)-(CTP)_4。将PCR获得的补体C3d-P28 DNA序列以头尾串连的方式构建四聚体。将(C3d-P28)_4基因片断克隆入pBSMR-(CTP)_4,得到pBSMR-(CTP)_4-(C3d-P28)_4,进一步得到表达质粒pET-28a(+)*(CTP)_4-(C3d-P28)_4。将表达质粒pET-28a(+)-(CTP)_4和pET-28a(+)-(CTP)_4- (C3d-P28)_4分别转化入大肠杆菌BL21(DE3),表达融合蛋白hCGβ-(CTP)_4和hCGβ-(CTP)_4-(C3d-P28)_4。采用Ni-NTA-resin和凝胶过滤方法纯化蛋白。结果:菌株经IPTG诱导后检测到目的基因的表达产物, SDS-PAGE和Western blot结果显示融合蛋白的分子量分别为27kD和40kD左右。结论:在BL21 (DE3)中成功表达了hCGβ-(CTP)_4和hCGβ-(CTP)_4-(C3d-P28)_4融合蛋白,并确立了纯化方法,为进一步研究其免疫原性和应用于抗肿瘤疫苗奠定了基础。Objective： To investigate the prokaryotic expression of the genes encoding proteins hCGβ-（CTP）4 and hCGβ-（CTP）4-（C3d-P28）4 and to purify the recombinant proteins. Methods： （CTP）4 coding gene from pBSMR-（CTP）4 was cloned into prokaryotic expression vector pET-28a（＋） and the expression plasmid pET-28a（＋）-（CTP）4was obtained. C3d-P28 coding gene was amplified by PCR. Four copies of the C3d-P28 gene were linked in tandem. Then （C3d-P28）4 was subcloned into pBSMR -（CTP）4 and pBSMR -（CTP）4-（C3d-P28）4 was obtained Finally, （CTP） 4-（C3d-P28）4 was subcloned into pET-28a （＋） and the expression plasmid pET-28a （＋）-（CTP）4- （C3d-P28）4was obtained. The plasmids pET-28a （＋）-（CTP）4 and pET-28a （＋）-（CTP）4-（C3d-P28）4were transformed into competent cell BL21 （DE3） respectively. The fusion proteins hCGβ-（CTP）4 and hCGβ-（CTP）4-（C3d-P28）4 were expressed in BL21 （DE3） and purified by Ni-NTA-resin and gel filtration. Results： After induced by IPTG, proteins were detected by SDS-PAGE and Western blot, which suggested that the proteins were expressed with molecular weights of about 27 kD and 40 kD. Conclusion： The recombinant proteins were successfully expressed in BL21 （DE3） and purified. This study provides necessary data for further investigation of the immunogenicity and anti-cancer vaccine of fusion protein hCGβ-（CTP）4-（C3d-P28）4.

关 键 词：hCGGβ-CTP多聚体 C3D 分子佐剂 表达 纯化 

分 类 号：Q789[生物学—分子生物学]