Construction And Appilication Of High-efficiency Expression Fowlpox Virus Vector

In an endeavor to enhance the level of foreign gene expression in recombinant fowlpox viruses(rFPVs), we constructed a high-effiency expression insertion vector on the basis of non-essential regions of FPV DNA and synthetic promoter (Ps) LLEE to transfer foreign genes into the FPV genome. This vector was derived through multiple molecular manipulations. The P7.5-P1 l-LacZ reporter gene cassette from plasmid pppGl8 was removed and inserted into Ps plasmid which bore a strong synthetic promoter LLEE, to let the Ps in opposite direction to P11 -LacZ to form pGS. The Ps-LacZ-P 11 cassete was removed and inserted into FPV7s to form insertion vector pFGS 11, which contained the Ps promoter to express foreign genes, the reporter gene LacZ under the control of the vaccinia virus (VV) P11 promoter in opposite direction to the Ps promoter and multipe cloning sites (BainHl, Sinai etc). For the construction of transfer vectors pFGBS and pFGFS, Ml)VgB gene of CV1988/Rispens strain in pMGB and NDV F gene of F48E8 strain in pF37 were removed and inserted into pFGS 11, downstream of Ps promoter, the two transfer vectors were confirmed by restriction endunuclease analysis and southern blot. Recombinant FPVs were constructed by DOSPER iiposome-mediated transfection with the two transfer vectors on chicken embryo fibroblast (CEF) 4 monolayer cultures which were infected with wild type FPV Chinese vaccine strain 282E4 3-4 hours earlier. Recombinant FPVs with blue plaques were selected and purified 6 times on secondary CEF subcultures overlaid with agarose containing X-gal. The expression of foreign genes in recombinant FPVs was confirmed by indirect immunofluoresence assay (WA) with specific monoclonal antibody. The differencial intensity of the synthetic promoter Ps and the vaccinia virus P7.5 promoter in recombinant FPVs was evaluated by using WA and Dot ELISA. The results showed that the level of foreign gene expression driven under the Ps promoter was three times higher than that driven under the P7.5 promoter. Therefore we constructed successfully a highly-efficient and universal expression vector.