Construction and expression of a recombinant adenovirus vector expressing the EAST-6-CFP-10 gene of Mycobacterium bovis.

A recombinant adenovirus vector expressing gene encoding the EAST-6-CFP-10 fusion protein of Mycobacterium bovis was constructed firstly by the amplification of the genomic DNA from M.bovis Vallee Ⅲ strain with PCR by gene SOEing method,then the EAST-6 and CFP-10 genes were linked with the(Gly4Ser)3 linker.The linked gene was inserted initially into vector pMD18-T and then subcloned to the adenovirus shuttle plasmid pShuttle-CMV to construct the recombinant shuttle plasmid pShuttle-CMV-EAST-6-CFP-10.It was confirmed correct by restriction endonuclease digestion,PCR and sequencing,lineared by Pme Ⅰ enzyme digestion and transformed into E.coli BJ5183-AD-1 containing plasmid pAdeasy-1 to form the homogeneous recombinant adenovirus plasmid vector.After the vector was identified by Pac I digestion and PCR,the lineared homogeneous recombinant plasmid was transfected to AD-293 cells using lipofectamineTM 2000.The fusion gene and its expression in virus were detected by PCR and Western blot.In these ways,the recombinant virus carrying the gene encoding the EAST-6-CFP-10 fusion protein was successively constructed,thus provide basis for the study on the immune effects in animals.