Construction, Optimization And Expression Of Mammalia Recombinant Vector For Truncated Fragment Of Human Osteoprotegerin

Osteoprotegerin(OPG), acts as the decoy receptor of receptor activator of nuclear factor- κB ligand (RANKL) and exerts its inhibitory effect on osteoclast differention and activation by binding RANKL. It is a secreted glycoprotein including a hydrophobic leader peptide, and 5 potential sits of N-linked glycasylation. It has 7 major structure domains, in which domains 1-4 (cysteinerich structures) are sufficient to inhibit osteoclastogenesis. Initially, OPG is synthesized as a monomer within the cell, is next converted to a dimmer linked by disulfide bond at Cys400 disulfide bond.The prokarotic expression of activated OPG protein is not successful until nowbecause there are some disadvantages. For example, the eukaryotic protein expressed in E. coli is not properly modified, without perfect N-linked glycosylation, phosphorylation, disulfide formation and so on, which make it difficult to obtain full native protein. The functional OPG protein was expressed in eukaryotic cells, but it is difficult to obtain sufficient amount of protein. Our laboratary has constructed human osteoprotegerin mammalia expression vector pcDNA3.1/DHFR-0PG that was primarily expressed in CHO cell, though the yield is not enough to satisfy continuous work. In the present study we improved the existing expression system and identified immunogenicity and determined bioactivity.In this research, the human truncated OPG cDNA including 21 signal peptides, domains 1-4 and Cys400 was amplified from the vector pcDNA3.1/DHFR-OPG by PCR, and then it was cloned into the EcoRI sit of the previously modified mannalia expression vector pcDNA3.1/DHFR-GFP. The recombinant construct was named pcDNA3.1/DHFR-tOPG. Then a try was made in which the recombinant vector was added with a 35bp enhancer and a 37bp intron at the 5' untranslated region of OPG by PCR, therefore obtaining a new construct named pcDNA3.1/DHFR-netOPG.In brief, the recombinant vector pcDNA3.1/DHFR-tOPG and pcDNA3.1/ DHFR-netOPG were respectively transfected into CHO- dhfr" cell and performed expression. The positive cells that expressed DHFR were screened by selective medium IMEM with 5% dialysis FCS. The higher-yield cell strains were determined by ELISA and cultured in gradient concentration MTX to screen the cell strains that can most efficiently and stably express recombinant protein. The expression yield of recombinant protein increased with the elevation of MTX concentration, the yield was up to 6ug/ml. there is no difference between pcDNA3.1/DHFR-tOPG and pcDNA3.1/DHFR-netOPG (p>0.05), Western Blotting identified the immunogenicityof recombinant human OPG with the molecular weight of about 37.8kD. The bioactivity analysis conformed the protein dose-dependently inhibited OLC development induced by RANKL and M-CSF. The expression of interest gene was reproducible after three freezing and thawing cycles (p>0.05).In conclusion, a higher efficient recombinant expression system was developed which produced a native and founctional form of human truncated OPG in the CHO-dhfr" cell.