Human recombinant amelogenin acquirement by using eukaryocyte expression system

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.