Cloning Of The GD, GE And TK Gene Of Pseudorabies Virus And Construction Of A Universal Transfer Vector

Three pairs of primers were designed according to the sequences pulished by the GeneBank in order to amplifiy gD,gE and TK gene of the pseudorabies virus Min-A strain.The gD,gE and TK gene were obtained by polymerase chain reaction(PCR), and then cloned into the pGEM-T Easy vector. These recombinant plasmids were identified by restriction enzyme analysis and sequencing ,Which proved completely their validity.Nucleotide sequencing revealed that the gD gene contained an open reading frame of 1203 bp encoding a 400 aa protein. The gD gene was subcloned into pcDNA3.1-,an eukaryotic expressing vector.The constructed expressing plasmid pcDNA-gD will be used for future gene vaccination.The gE gene contained an open reading frame of 1740 bp encoding a 579 aa protein. The homology of the nucleotide sequence of PRV Min-A strain gE with PRV Ea strain,SH strain was 99.2%, 98.7%,respectively ;and the homology of predicted amino acids among them was 98.6%, 97.2%, respectively. The TK gene contained an open reading frame of 957 bp encoding a 318 aa protein.Upstream of the TK ORF,three putative GC boxes are located at positions -22,-166 and -199. A potential poly A signal begins 110 nucleotides downstream from the termination codon at position 1306.The homology of the nucleotide sequence of PRV Min-A strain TK with PRV Ea strain,NIA-3 strain was 98.4%, 97.9%,respectively ;and the homology of predicted amino acids among them was 97.8%, 97.2%, respectively. We have identified six conserved domains by multiple sequence alignments of the thymidine kinase proteins of the alphaherpesviruses. It is speculated that these conserved ammo acidresidues is necessary for structural stability or involved in functional domains responsible for catalytic activities.The gD and gE gene was subcloned into pUC18,resulting in pUgDgE.The fragment from pcDNAS.1- including hCMV promoter/enhancer,MCS and neomycin resistance gene was inserted into the BamHI and BstEII restrication sites of pUgDgE,resulting in the universal transfer vector pgD-M-gE.The universal transfer vector pgD-M-gE has deleted the gI gene and 363bp in the 5'end of the gE ORF of PRV.There were 11 restrication sites for insertion of the foreign gene.The upstream and downstream flanking sequences were up to 1.25kb and 1.42kb.It will be useful for developing the recombinant PRV expressing foreign gene(s).