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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。
Objective To construct a recombinant eukaryotic expression plasmid containing human amelogenin (hAm) gene and transfect mammalian cell line NIH3T3 to establish a cell line stably expressing recombinant hAm, which will lay the foundation for clinical application. Methods The hAm gene was inserted into the eukaryotic expression vector pcDNA3.1 / myc-His (-) A by restriction endonucleases EcoRⅠ and BamHⅠ to construct the recombinant plasmid pcDNA3.1 / myc-His (-) A-hAm , Double digestion and sequencing identification. NIH3T3 cells were transfected with LipofectamineTM2000-mediated recombinant plasmid, and positive clones were screened by G418. Stably expressing human amelogenin cell lines were stained with SDS-PAGE and Western blotting. Results The recombinant plasmid pcDNA3.1 / myc-His (-) A-hAm was verified by double enzyme digestion and sequencing. The recombinant plasmid was transfected into NIH3T3 cells and the stable transfected cell lines were confirmed by SDS-PAGE electrophoresis and Western blotting. The relative molecular mass of hAm was 28 000, consistent with the prediction. Conclusion The recombinant eukaryotic expression system containing hAm gene was successfully constructed and NIH3T3-hAm cell line stably expressing recombinant hAm was obtained.