Transcriptional activity comparison of different sites in recombinant Marek's disease virus for the expression of the H9N2 avian influenza virus hemagglutinin gene

Over the last two decades, much attention has been paid to MDV-vectored recombinant vaccines. Many factors have influenced their protective efficacy, and insertion site has been among the main influential factors for the expression of foreign genes in recombinant Marek's disease virus (rMDV). To compare the transcriptional activity of different sites of rMDV, an H9N2 avian influenza virus hemagglutinin gene (AIV-H9N2-HA) expression cassette that used the bi-directional promoter of serotype 1 MDV (MDV1) in the 1.8 kb RNA transcript direction (p1.8 kb) as a promoter was inserted into 4 different regions of MDV using the bacterial artificial chromosome (BAC) vector and FLP/FRT recombination technique. The insertion regions included 3 of its own sites (US2, US10 and one of Meq genes) in the MDV genome and a foreign site (gpt gene) in the BAC vector. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to analyze and compare the H9N2-HA expression levels of these different rMDVs both at the mRNA level and at the protein level. The results indicated that among the four tested insertion regions, the HA expression cassette in the US2 region demonstrated the highest activity, followed by that in the Meq region, which was almost equal to that of US10. Further, the expression cassette had the lowest activity in the foreign region gpt gene. The above data could be useful for choosing proper recombinant insertion regions in the construction of rMDV to express different foreign genes, and it is a prerequisite for developing effective MDV-vectored recombinant vaccines.