[Construction of the stable cell line expressing the mouse recombinant enamelin gene].
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.


CONCLUSIONS
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.
Lou
Ya li
Yl
Department of Prosthodontics, Peking University School & Hospital of Stomatology, Beijing 100081, China.
Sun
Ying lin
Yl
Zhao
Tie qiang
Tq
Liu
Guo qing
Gq
Feng
Hai lan
Hl
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D003001:Cloning, Molecular; D003746:Dental Enamel Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D003746:Dental Enamel Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C038456:tuftelin