Construction and Selection of Strong Promoter for Fowlpox Virus Vector
For constructing strong combined promoter to enhance the foreign gene expression in Fowlpox virus(FPV) vector, four oligonucleotides were synthesized according to the essential structure of the vaccinia virus nature promoter P7.5 and were used for generating early and late promoters. Ten artificial promoters designated PS1～10 were constructed by using different combination of early and late promoters, which were recognized by restriction endonuclease analysis and sequence analysis. LacZ gene transfer vectors of FPV with these promoters and P7.5 were generated by a series of molecular biology manipulation. DOSPER liposome mediated transfection with these transfer vectors was performed on chicken embryo fibroblast (CEF) monolayers, which was infected with FPV Chinese vaccine strain 282E4 3～4 hours earlier, to produce recombinant viruses. Recombinant FPVs with blue plaque were selected and purified 4 times on secondary CEF subcultures overlaid with agarose containing X Gal. The level of the β galactosidase expressed by these recombinant viruses with LacZ gene under the control of various synthetic promoters and P7.5 were assayed and compared in the presence or absence of cytosine arabinoside (Arc C) at 10, 24, 36, 48, 72 hours post infection respectively. The LLEE combination was the strongest synthetic promoter selected out which drived expression of β galactosidase three more times higher than the P7.5, and appeared to be valuable for the construction of more efficient expression FPV vector.