The Construction Of Recombinant Adenoviral Vector Overexpressing Betatrophin

Objective To construct an replication deficient recombinant adenoviral vector expressing mouse betatrophin gene by using gene homologous recombination and to provide an experimental basis for further studying the function of betatrophin.Methods From the sequence of Gen Bank, betatrophin gene including of Bam H I/Age I restriction enzyme cutting sites was obtained by chemical synthesis and was inserted into the GV314 vector(CMV-MCS-3FLAG-SV40-EGFP) carrying enhanced green fluorescent protein(EGFP) to construct shuttle plasmid p GV314-betatrophin. Using Ad-Max packaging system,The shuttle plasmid GV314-betatrophin and adenovirus genomics plasmid p BHG loxΔE1, 3 Cre were cotransfered into 293 T cells by using gene homologous recombination in vitro to construct recombinant adenovirus vector Ad-betatrophin. The DNA of recombinant adenovirus vector was finally transfered into HEK293 T cells to package recombinant adenovirus particles. Recombinant adenovirus particles were repeat amplification in 293 T cells to check and identify supernatant of adenovirus by PCR, western blot and sequencing technologies.Results(1) The target gene betatrophin was obtained by chemical synthesis, and the product was identified correctly by digestion.(2) The plasmid pc DNA3.1-betatrophin was digested by Bam H I/Age I restriction enzyme and inserted into the GV314 vector by Infusion cloning technique, the product is the shuttle plasmid GV314-betatrophin and was successful constructed by PCR, western blot and DNA sequencing.(3) The shuttle plasmid GV314-betatrophin and adenovirus genomics plasmid p BHG loxΔE1, 3 Cre were cotransfered into 293 T cells. The recombinant adenovirus Ad-betatrophin were packaged successfully.(4) The recombinant adenovirus p Ad-betatrophin gene transferred into HEK293Tcells were packaged successfully. After being amplified and purified, the titer of recombinant adenovirus was 1×1010.2pfu/ml.Conclusion The recombinant adenovirus vector carrying mouse betatrophin gene was successful constructed to further explore the function of betatrophin.