中华口腔医学研究杂志(电子版) ›› 2008, Vol. 02 ›› Issue (02) : 135 -140. doi: 10.3877/cma.j.issn.1674-1366.2008-02-006 × 扫一扫
基础研究
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Ying-lin SUN1, Wei HUANG1, Guo-qing LIU1, Hai-lan FENG1,†()
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孙樱林, 黄薇, 刘国庆, 冯海兰. 通过真核细胞表达系统获得重组人釉原蛋白[J/OL]. 中华口腔医学研究杂志(电子版), 2008, 02(02): 135-140.
Ying-lin SUN, Wei HUANG, Guo-qing LIU, Hai-lan FENG. Human recombinant amelogenin acquirement by using eukaryocyte expression system[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2008, 02(02): 135-140.
目的
构建重组人釉原蛋白基因的真核表达系统，并建立稳定表达该蛋白的细胞系。
方法
取26 周龄引产胎儿的牙胚组织，提取总RNA，用RT-PCR 技术扩增釉原蛋白基因片段，插入中间表达载体pGEM® -T。 经双酶切后，再与真核表达载体pcDNA3.1TM/myc-His（-）B 相连接，构成最终的表达质粒，将该重组表达质粒转染至HEK 293A 细胞，用G418 筛选出阳性细胞克隆，并建立稳定表达釉原蛋白的细胞系。
结果
通过测序表明，人釉原蛋白基因被成功地连接到了真核表达载体上。 将该表达系统转染HEK 293A 细胞后，进行Western Blot 检测，证明有相对分子质量约32 000 的釉原蛋白表达。
结论
本实验成功构建了重组人釉原蛋白真核表达系统，建立了稳定细胞系，为获得高纯度的釉蛋白，进一步研究蛋白质功能奠定了基础。
Objective
The objective of this study was to construct a human recombinant amelogenin eukaryocyte expression system， and establish a stable cell line which can produce this protein continuously.
Methods
mRNA transcript was extracted from the teeth germ of a 26-week preborn. The amelogenin gene fragment was amplified with RT-PCR technique. The PCR product was cut with two restriction enzymes， and subcloned into the eukaryotic gene expression vector pcDNA3.1TM/myc-His（-）B. The recombinant expression plasmid was transferred into HEK 293A eukaryocyte cells. G418 was used to selectively culture the cells and scan the positive cell clones. HEK 293A cell line expressing human recombinant amelogenin was successfully established confimed by western blot analysis.
Results
The human amelogenin gene was cloned into the eukaryotic expression plasmid successfully by verification of sequence measuring. After transfection of the recombinant plasmid into the HEK 293A cells， about 32 000 Da amelogenin protein was detected by SDS-PAGE and Western Blot.
Conclusions
A recombinant eukaryocyte expression plasmid and a stable cell line were established， laying a foundation for obtaining biologically active amelogenin protein with high purity and investigating its function further more.