[Construction of mammalian expression plasmid of Flag-tagged rat collagenase and its in vitro transfection study].
OBJECTIVE
A mammalian expression plasmid of rat collagenase was constructed and its expression in NIH3T3 cells in vitro was studied.


METHODS
PCR and gene recombinant techniques were used to construct a mammalian expression plasmid of Flag-fusion rat collagenase. This plasmid was then transferred into cultured NIH3T3 cells mediated by lipofectamine. The mRNA and protein expression of the Flag epitope tagged rat collagenase were detected by RT-PCR and western blot techniques. The enzyme activity of expressed collagenase was detected by gelatin zymography.


RESULTS
After transfection the mRNA expression of Flag-fusion rat collagenase could be detected in NIH3T3 cells and the secretion of this collagenase could be detected in the culture medium, and this recombinant collagenase kept the gelatin degradation activity.


CONCLUSIONS
This constructed plasmid can express active rat collagenase in vitro and can be used for further study.
Guo
J
J
Department of Gastroenterology, Zhongshan Hospital, Shanghai Medical University.
Wang
J
J
D016475:3T3 Cells; D000818:Animals; D003094:Collagen / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D051381:Rats; D014162:Transfection
D003094:Collagen