Cloning And Sequence Analysis Of The TK Gene Of Pseudorabies Virus JA Strain And Construction Of TK～- Strain Transfer Vector

An analysis of homology of the nucleotide sequence and its deduced amino acid sequence with other strain which published by the GeneBank. Using the genome DNA of the strain JA of pseudorabies virus as template, the TK gene fragment was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pUC 19 plasmid vector and the nucleotides of the TK gene was sequenced. Resulting in the homology of the nucleotide sequence and amino acid sequence of JA TK ORF with other strain was upwards of 99%. Upstream of the initiation codon, three putative GC boxes are located. A potential poly A signal was detected at downstream from the termination codon. We have identified conserved domains of the thymidine kinase proteins of the alpha herpes viruses:—R*Y*DG**G*GK*T—and—FDRHP*A***C*P*AR—.In this text, TKL and TKR were amplified from TK gene of strain A, PRV as homologous arms for shuttle plasmid. And then they were cloned into vectors pUC19和pBluescript SK for recombinant plasmid. Positive clones were selected using enzyme digestion. And the result showed that there was the highest nucleotide homology between genes TKL, TKR in the recombinant plasmid and strain NIA published on Genebank, 96.2% and 93.6% respectively; Compared with other strains, UL24 of strain A, PRV shows one more enzyme digestion site BamHI due to its insertion mutation (A) on site 1347.For the purpose of selecting and identificating recombinant plasmid, lacZ was inserted as a report gene. LacZ expressing cassest with promoter for PRV gG was transformed after digestion plasmid TT80 by SalI and BamHI. In this text, subcloning was used to ligate sticking end ligate to enhance efficancy. Firstly, plasmid pBSK_lacZ was aquired by cloning LacZ expressing cassest into pBluescript_SK and then pBSK_lacZ was digested by SalI and BamHI and was cloned into plasmid pUC_TKL to get recombinant plasmid pUC_TKL_lacZ. At last, TKR was digested by XbaI and KpnI and was cloned into plasmid pUC_TKL_IacZ to get transfer vector pUC_TKL_lacZ_TKR.