The Culture And Identification Of Cos-7 Cell Lines Modified By Osteoprotegerin Gene

Objective: In order to understand the ability of expression and secretion of eukaryotic expression plasmid of human osteoprotegenrin, for preparing gene engineering therapy of periodontitis, we constructed this plasmid and detected its expression in Cos-7 cell lines in this study.Method: The oligonucleotide primers were designed and synthesized based on the human OPG cDNA sequence. Total mRNA was isolated from 293 cells and RT-PCR was performed. The fragment was inserted into pSecTag2/B vector and sequenced by automatic sequence analyzer. The constructed recombined plasmid was transformed into Cos-7 cell line and OPG expressed in Cos-7 cell line was determined by immunohistochemistry and western blot.Result: The sequence of OPG cDNA from 293 cell by RT-PCR was completely identical to the sequence provided by GenBank [gi:33878056]. The recombinant plasmid digested with Hind III, EcoR I and BamH I,10g/L agoarose electrophoresis showed several fragments, which were consistent with predicted size. Recombinant plasmid pSecTag2/B-OPG transformed into Cos-7 cell line, OPG over-expression Cos-7 cell line was selected and confirmed by immunohistochemistry and western blot.Conclusion: pSecTag2/B-OPG vector has been constructed successfully and OPG over-expression in Cos-7 cell line was determined.