Abstract: To develop duck enteritis virus （DEV） and duck Tembusu virus （DTMUV） bivalent vaccine， the recombinant plasmid pEP\|BGH\|E （containing expression cassette pCMV\|E\|BGH\|pA） was constructed by inserting codon\|optimized DTMUV E gene into the expression vector pEP\|BGH\|end. The BAC clone of pDEV\|E was generated by twice Red/ET recombination in E. coli based on the bacterial artificial chromosome （BAC） clone pDEV\|vac which carried whole DEV genome. Firstly， the BAC clone pDEV\|EF1 was constructed by replaced CMV promoter of GFP gene with EF1 promoter on pDEV\|vac; then pDEV\|E was constructed by inserting pCMV\|E\|BGH\|pA between DEV US7 and US8 gene on pDEV\|EF1. The recombinant virus rDEV\|E was rescued from chicken embryo fibroblasts （CEFs） transfected with pDEV\|E by calcium phosphate precipitation. The plaque size of rDEV\|E was slightly decreased compared with the parental virus rDEV\|BAC. Western blotting analysis showed that DTMUV E protein was expressed in rDEV\|E virus\|infected CEFs.

Key words: duck enteritis virus, duck Tembusu virus, E protein, bacterial artificial chromosome, recombinant virus