Antibody Preparation And Localization Of The UL24 Protein Of Pseudorabies Virus

Pseudorabies virus (PrV) is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals. PrV infection could cause its natural and main storing host-swine (also its main infectious origination) a huge loses, the most huge economical lose in all swine infectious diseases except Classic Swine Fever and Food and Mouth Disease.A pair of primers flanking the UL24 gene was designed according to the published PrV nucleotide sequence. The UL24 gene of the PrV Rong A stain was abtained by polymerase chain reaction (PCR) and was cloned into pEGFP-N1 vector and the nucleotides of the UL24 gene was sequenced. The sequencing result showed that the UL24 gene of PrV Rong A strain contain a single open reading frame capable of encoding 171 amino acids. By sequence analysis with DNA Star software, we found that the UL24 gene was very conserved between PrV strains and also conserved among herpesvirus, and it had five conserved motifs which may be it's structural or functional domains.According to PrV UL24 nucleotide sequence published on GenBank a pair of specific primers were synthesized to amplify the modified UL24 (mUL24) with PCR method from recombinant plasmid pUL24-GFP. The mUL24 was absent of the dense section of rare codons for E.coli in the UL24 gene's 3' and 5' terminal on the precondition of making no difference to coding sequence to enhance the-level of expression. The PCR product degested with BamH I and Hindi III and then cloned into pET-32a(+) vector to get a prokaryotic recombinant plasmid pET-UL24. Transformed pET-UL24 plasmid into BL21(DE3) host cell and the recombinant protein (His)6-UL24 was successfully expressed by the form of infusion body when induced with IPTG. Target protein was purified with the Ni²⁺-NTA His-Bind(?) Purification Kit . The purified protein immuned rabbits and the antibody was getted when the serum antibody level had reached tol :2560. The result of this experimentation lay the foundations of the intracellular localization study of pseudorabies virus UL24 protein.