Cloning And Sequence Analysis And Construction Of Prokaryotic Expression Vector Of The UL17 Gene From Porcine Pseudorabies Virus FZ Strain

Pseudorabies (PR) has become one of the most dangerous disease of swine resulting in devastating economic losses worldwide. While efforts to eradicate PR in the United States and Europe have shown great progress, it is still an endemic problem in many countries. This herpesvirus has served as a useful model organism for the study of herpesvirus biology and become an increasing concern. PRV UL17 gene is 1736bp in length, encoded 597 amino acids, the molecular weight is 64kDa. The UL17 protein is a component of the viral capsid , and required for DNA cleavage and packaging in PRV . In this study , we cloned and constructed the prokaryotic expression vector of the UL17 gene of PRV FZ strain which is isolated, identified and preserved in our laboratory.According to the published sequence of PRV UL17, a pair of specific primers was designed to amplify the UL17 gene from the viral DNA, and this amplified production was cloned into pMD-18T Simple Vector and sequenced. Owing to the high contents of G+C, the result of sequencing showed that it has been detected 1736 bp in length, about 60bp were failed. This sequence was compared with other published sequence of UL17 by DNAStar. The results showed that the sequences of our study were closely related to those of other two strains, Ea and Ka, in the GenBank, and homology of UL17 sequence were more than 96%. Conserved Domain Search revealed that the UL17 protein contains two conserved domains. The amino acid sequence analysis shows that Protein Kinase C phosphorylation site and casein kinase II phosphorylation site are found in the acid sequences of UL17 gene, which suggests that UL17 gene is a possible phosphoprotein.Insert the UL17 gene fragment into the prokaryotic expression vector pET-32a(+), named pET-UL17, and was transferred to E.coli DH5_α. Successful transformation was confirmed both by PCR amplification and restriction enzyme digestion. Codons bias analysis showed that UL17 biased in some codons, the amino acid composition is skewed to codons rich in Gs and Cs nucleotides, especially the third codon is Cs or Gs.