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[Construction of the stable cell line expressing the mouse recombinant enamelin gene]
Ya li Lou1, Ying lin Sun, Tie qiang Zhao
1Department of Prosthodontics, Peking University School & Hospital of Stomatology, Beijing 100081, China.
Objective:
To construct a mouse recombinant enamelin eukaryocyte expression system, and establish the stable cell line which can produce the protein continuously.
Methods:
The mRNA transcript from the 3-day mouse jaw was extracted. and the enamelin gene fragment amplified with RT-PCR techniques. Then the PCR product was cat with two restriction enzymes, and subcloned into the eukaryotic gene expression vector pcDNA3.1TM/mycj His(-)B.The recombinant plasmid was transformed into E.coli DH5alpha bacterial cells, and harvested with plasmid midi kit. The recombinant expression plasmid was transferred to the HEK 293A eukaryocyte cells, cultured selectively with 800 mg/L G418, and examined with SDS-PAGE and Western Blot at the protein level.
Results:
The mouse enamelin gene was cloned to the eukaryotic expression plasmid successfully by sequence measuring. After the recombinant plasmid was transferred into the HEK 293A cells, about 32,000 enamelin protein was checked out by SDS-PAGE and Western Blot.
Conclusion:
The recombinant eukaryocyte expression plasmid and the stable cell line were established. This is a basic research to obtain high-yeild biologically active enamelin protein, which may facilitate further investigation of its function.
