上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (8): 1108-.doi: 10.3969/j.issn.1674-8115.2011.08.014

• 论著（基础研究） • 上一篇    下一篇

重组人釉原蛋白真核表达载体的构建及其在NIH3T3细胞中的稳定表达

程 岚1, 束 蓉1, 张秀丽1, 田 聆2   

  1. 1.上海交通大学 医学院附属第九人民医院牙周科 上海市口腔医学重点实验室, 上海 200011； 2.上海交通大学附属第一人民医院实验中心, 上海 200080
  • 出版日期:2011-08-28 发布日期:2011-08-29
  • 通讯作者: 束 蓉, 电子信箱: shurong123@hotmail.com。
  • 作者简介:程 岚（1978—）, 女, 主治医师, 博士生；电子信箱: chenglan920@126.com。
  • 基金资助:

    国家自然科学基金（81070838）

Construction of recombinant human amelogenin eukaryon expression vector and its stable expression in NIH3T3 cells

CHENG Lan1, SHU Rong1, ZHANG Xiu-li1, TIAN Ling2   

  1. 1.Department of Periodontology, the Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China；2.Laboratory Center, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China
  • Online:2011-08-28 Published:2011-08-29
  • Supported by:

    National Natural Science Foundation of China, 81070838

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摘要：

目的 构建含人釉原蛋白(hAm)基因的重组真核表达质粒并转染哺乳动物细胞NIH3T3，建立稳定表达重组hAm的细胞株，为临床应用奠定基础。方法 利用限制性内切酶EcoRⅠ和BamHⅠ将hAm基因插入真核表达载体pcDNA3.1/myc-His(-)A，构建含hAm基因的重组质粒pcDNA3.1/myc-His(-)A-hAm，双酶切和测序鉴定。通过LipofectamineTM2000介导重组质粒转染NIH3T3细胞，利用G418筛选出阳性克隆，建立稳定表达人釉原蛋白的细胞株，聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blotting验证蛋白表达。结果 重组质粒pcDNA3.1/myc-His(-)A-hAm经双酶切和测序鉴定证实插入序列准确无误。重组质粒转染NIH3T3细胞后建立的稳定转染细胞株经SDS-PAGE电泳及Western blotting检测显示有相对分子质量为28 000的hAm表达，与预测一致。结论 成功构建含hAm基因的重组真核表达系统，并获得稳定表达重组hAm的细胞株NIH3T3-hAm细胞株。

关键词: 重组人釉原蛋白, 真核表达载体, 转染, 稳定表达

Abstract:

Objective To construct the recombinant eukaryon expression plasmid containing human amelogenin (hAm) gene and transfect mammalian cell line NIH3T3 for construction of cells with stable expression of recombinant hAm. Methods hAm gene was inserted into eukaryon expression vector pcDNA3.1/myc-His (-) A with restriction enzyme EcoRⅠ and BamHⅠ, and recombinant plasmid pcDNA3.1/myc-His (-) A-hAm containing hAm gene was confirmed by restriction  endonuclease mapping and sequencing. pcDNA3.1/myc-His (-) A-hAm was transfected into NIH3T3 cells by LipofectamineTM2000, and was selected by G418 for positive cell clones. Cells with stable expression of hAm was constructed, and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results Restriction endonuclease mapping and sequencing revealed that the inserted sequences were accurate in recombinant plasmid pcDNA3.1/myc-His (-)A-hAm. Expression of hAm with molecular weight of 28 000 was detected by SDS-PAGE and Western blotting in NIH3TS cells transfected with recombinant plasmid, which was in line with the prediction. Conclusion The recombinant eukaryon expression system containing hAm has been successfully constructed, and NIH3T3 cells with stable expression of recombinant hAm is obtained.

Key words: recombinant human amelogenin, eukaryon expression vector, transfection, stable expression