| hCGβ-(CTP)_4和hCGβ-(CTP)_4-(C3d-P28)_4在大肠杆菌中的表达及纯化 |
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| 引用本文： | 石燕,蒋俶,沈卫英,蒋雅红,申庆祥.hCGβ-(CTP)_4和hCGβ-(CTP)_4-(C3d-P28)_4在大肠杆菌中的表达及纯化[J].生殖与避孕,2007,27(4):241-246,279. |
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| 作者姓名： | 石燕 蒋俶 沈卫英 蒋雅红 申庆祥 |
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| 作者单位： | 1. 复旦大学上海医学院,上海,200032;上海市计划生育科学研究所,上海,200032
2. 上海市计划生育科学研究所,上海,200032 |
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| 基金项目： | 国家自然科学基金;上海市科技攻关项目 |
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| 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。
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| 关 键 词： | hCGβ-CTP多聚体 C3d 分子佐剂 表达 纯化 |
| 文章编号： | 0253-357X（2007）04-0241-06 |
| 修稿时间： | 2007-02-14 |
Expression and Purification of the Fusion Proteins hCGβ-(CTP)4 and hCGβ(CTP)4-(C3d-P28)4 in E.coli |
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| Yan SHI,Shu JIANG,Wei-ying SHEN,Ya-hong JIANG,Qing-xiang SHEN.Expression and Purification of the Fusion Proteins hCGβ-(CTP)4 and hCGβ(CTP)4-(C3d-P28)4 in E.coli[J].Reproduction and Contraception,2007,27(4):241-246,279. |
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| Authors: | Yan SHI Shu JIANG Wei-ying SHEN Ya-hong JIANG Qing-xiang SHEN |
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| Institution: | 1. Shanghai Medical College, Fudan University, Shanghai, 200032;2. Shanghai Institute of Planned Parenthood Research, Shanghai, 200032 |
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| Abstract: | 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)_4coding 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 trans- formed 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 27kD and 40kD.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. |
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| Keywords: | C3d |
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