| Objective To construct an recombinant adenovirus plasmid of SEDLgene, and generating high titer recombinant adenovirus. To enhance theexpression of SEDL mRNA and sedlin in infected hFOB1.19cells usingthe recombinant adenovirus (Ad5-SEDL), which lays a real goodfoundation for the role of the SEDL gene and sedlin protein in bonemetabolism.Methods The specific SEDL gene was synthesized in vitro, andsubcloned into the shuttle plasmid pAdrrace-TO4, named afterpAdrrace-SEDL. Then cotransfecting with adenovirus skeleton plasmidpAdeasy-1to produce the recombinant adenoviral plasimid pAd5-SEDL byhomologous recombinantion.The pAd5-SEDL was identified by enzymecutting. We transfect pAd5-SEDL into package cell HEK293by PolyJet and generate high titer recombinant adenoviral Ad5-SEDL by usingping-pong infection.The hFOB1.19cells were infected with recombinantadenovirus Ad5-SEDL and divided into two groups: hFOB1.19/Ad5-SEDLgroups, and hFOB1.19groups. The SEDL mRNA and sedlin protein expression were determined by RT-PCR and Western blot. Cellproliferation was measured by MTT assay of hFOB1.19cells.Results (1) The recombinant adenovirus plasmid pAd5-SEDL wasconstructed successfully by enzyme cutting and gene screening.(2) Therecombined adenoviral plasimid pAd5-SEDL was transfected into thepackage cell HEK293successfully.(3) The high titre of the recombinantadenovirus pAd5-SEDL was4.3×l011pfu／ml.(4) SEDL mRNA and thesedlin protein were detected in all two groups.(5) The levels of mRNA ofSEDL in hFOB1.19/Ad5-SEDL group were（0.79±0.12） which weredecreased significantly than that in the group of hFOB1.19（0.11±0.04）(p<0.01).(6) The levels of sedlin protein were（0.93±0.30）in hFOB1.19/Ad5-SEDL group, which were higher than that in another group（0.12±0.04）(p<0.01).Conclusion (1) The recombinant adenovirus plasmid expressing theSEDL gene was constructed and generated high titer recombinantadenovirus which infected hFOB1.19cells successfully.(2) The hFOB1.19cells could express SEDL mRNA and the protein of sedlin.(3)Therecombinant adenovirus Ad5-SEDL could enhance SEDL mRNA andsedlin expression efectively in hFOB1.19cells. |
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