Construction of the stable cell line expressing the mouse recombinant enamelin gene

Objective: To construct a mouse recomb inant enamelin eukaryocyte expression system, and establish the stable cell line which can produce the protein continuously. Methods: The mRNA transcrip t 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 tw o restriction enzymes, and subcloned i nto the eukaryotic gene expression vector pcDNA3.1TM/myc-His(-)B. The recombinant plasmid was transformed into E.coli DH5α bacterial cells, and harv ested with plasmid midi kit. The recombinant expression plasmid was transferred to the HEK 293A e ukaryocyte 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 measurin g. 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. C o nclusion: The recombinant eukaryocyte expression plasmid and the stable cell line were est ablished. This is a basic research to obtain high-yeild biologically active ena me lin protein, which may facilitate further investigation of its function.