Background Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRV Δ TK&gE−AH02 ) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRV TK&gE−AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. Results In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection ( P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE ( P = 0.097) and PRV-S(UL46-27)ΔTK/gE ( P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRV Δ TK&gE−AH02 ). Conclusions This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.
【저자키워드】 spike gene, bacterial artificial chromosome, insertion site, Pseudorabies virus, Noncoding region, 【초록키워드】 Vaccine, Gene Expression, Vaccines, antibody, Genome, variant, virus, PCR, Real-time PCR, mutant, immunofluorescence, expression, recovered, Bacterial, S gene, BAC, Safe, epidemic diarrhea, chromosome, growth, insertion, mRNA expression, virulent, post infection, virus strain, Affect, Cell, Result, shown, exhibited, parental, expression cassette, Pseudoraby, 【제목키워드】 identification, insertion, Virus vector,