Amplification and cloning of Marek's disease virus glycoprotein D gene by polymerase chain reac- tion

arek's Disease Virus(MDV)glycoprotein D(gD) gene was amplificated from CEF ge-nomic DNA infected with MDV GA strain by polymerase chain reaction(PCR).PCR products ofthe gD gene were labeled with digoxigenin as porbes and they could only detect MDV-CEF ge-nomic DNA,MDV genomic DNA Bam HI-A fragment DNA by dot Not,but not uninfected CEFDNA,PCR products of the gD gene were cloned into pUC 18 plasmid,and recombinant plasmidswere screened by in situ hybridization with the probes of gD PCR products.The recombinantplasmids were also labeled with digoxigenin as probes.Two probes could detect gD PCR products,insert of recombinant plasmids,MDV genomic DNA BamHI-A fragment DNA by cross-hybridiza-tion in Southern blot.Restriction endonucleases analysis was used to identify the restriction sitesof the gD gene and polylinker of recombinant plasmid,and it showed that there were no sites of EcoRI、KpnI、HindⅢ、PstⅠ、Sma Ⅰ and PvuⅡ.It demonstrated that the recombinant plasmidcloned from MDV GA strain was gD clone.