通过真核细胞表达系统获得重组人釉原蛋白

Human recombinant amelogenin acquirement by using eukaryocyte expression system

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HEK293A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HEK293A细胞后,进行Western Blot检测,证明有相对分子质量约32000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 pcDNA3.1^TM/ myc-His（-）B. The recombinant expression plasmid was transferred into HEK 293A eukaryocyte ceils. 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.