B Cell Epitope Prediction And Construction Of A Bacterial Artificial Chromosome Of Variant Swine Pseudorabies Virus

Swine pseudorabies, caused by pseudorabies virus(PRV), is an acute infectious disease causing reproductive disorder in sows and acute death in piglets. PRV belongs to the family Herpesviridae, subfamily Alphaherpesvirinae. The mature vrions are composed of four unique structures: the core of double stranded linear DNA, icosahedral nucleocapsid, tegument, and outer envelope. The genome of PRV is about 140 kb containing 74% GC and at least includes 70 genes which encode about 100 proteins.Variant pseudorabies viruses have caused severe infections in many large pig farms on which pigs had been vaccinated with attenuated vaccines generated from the vaccine strain Bartha-k61 since 2011. To get a better understanding of the molecular basis of those variant strains, multiple sequence alignment and phylogenetic evolutionary analysis were performed on the protein sequence of gD, gB, gC, gH, gM, gL, gK, gN, UL34 and UL43 of four Chinese PRV isolates and the reference strains. B cell epitopes in those proteins were predicted using online tools BepiPred1.0b and Bcepred. Results showed that the four Chinese isolates clustered in separate branches from the seven foreign reference isolates in the phylogenetic trees generated with protein sequence of gD, gB, gC, gM, gK, gN,UL34,and UL43 of. The isolate HeN1 was, however, alone in the gL and gH trees, while the rest three Chinese isolates grouped with Becker and Bartha in these two trees, respectively. Compared with the Bartha strain, there are 31 predicted epitopes showing amino acid differences in the 11 envelope proteins of those Chinese isolates and these may be contributed to their antigenic variations.To construct a Bacterial Artificial Chromosome(BAC) containing the full genome of the vanriant PRV for the study of its antigenic and pathogenic mechanisms, we amplified homologous arms from the genomic DNA of a variant PRV strain HLJ by PCR and the EGFP gene, then they were ligated into the plasmid pUC18 by homologous recombination and the BAC were inserted into the combined vector. The transfer vector and the full length of genome disposed with the Acclâ… were co-transfected into BHK21 cell to generate the recombinant virus.The recombinant virus was identified using PCR, electron microscope observation and growth curve. The results showed that the BAC contains the complete encoding gene of HLJ strain, the green fluorescence can be expressed steadily, and the recomninant virus has similar morphology and growth cure with the wild type virus. These indicates that the BAC of variant PRV strain has been successfully constructed.