Gene Cloning and Prokaryotic Expression of Human Cytomegalovirus Glycoprotein gB/AD-1

Objective To clone the gene fragment gB/AD-1 encoding the glycoprotein of human cytomegalovirus(HCMV) and construct its prokaryotic expression system.Methods Amplify gB/AD-1 gene fragment from the genomic DNA of HCMV AD169 strain and clone into vector pGEM-T,then,after identification by sequencing,subclone to expression vector pET-15b. Transform the constructed recombinant plasmid pET-15b/gB/AD-1 to E.coli for expression of gB/AD-1 under induction of IPTG.Purify the expressed product by one-step immobilized metal ion affinity chromatography(IMAC) and identify by Western blot.Results The expressed gB/AD-1 contained 35% of total somatic protein.The purity of expressed product after purification was more than 95%.Western blot showed specific reaction of the expressed product with CMV antibody-positive human serum.Conclusion The recombinant plasmid pET-15b/gB/AD-1 was successfully constructed,and gB/AD-1 was highly expressed in E.coli.