GM-CSF (granulocyte-macrophage colony stimulating factor) is one of cytokines produced by immune effector cells in the earliest stage, participating in the regulation of the immune response in humans, animals including chickens. GMCSF, if administered as a biological adjuvant, will enhance the body to regulate the immunosystems working well against infectious diseases. Adenovirus HAd5 system (Stratagene) was chosen for construction of a recombinant adenovirus vector haboring the cytokine gene chGMCSF, for enhancement of immunity in poultry. First of all, the cytokine cassette KZ-chGMCSF was generated from the PCR product (size of 462 bp, amplified by CHF-CHR specific primerpairs). This cytokine cassette then was clonned into the pShuttle-CMV plasmid to construct a recombinant vector, namely, pShut-CMV-KZ-chGMCSF. This vector was transfected by the method "subsequent homologous recombination", into E. coli BJ5183 cells previously haboring the pAdeasy-l vector (namely, E. coli-BJ5183-pAdeasy-l), in order to facilitate homologous recombination of these two DNAs for generation of recombinant adenoviralplasmid, as pAdpShuttle-KZchGMCSF. Screening this recombinant plasmid was carried out by culturing recombinant E. coli cells in a kanamycine-containing medium. Selection of positive recombinant plasmid by restriction enzyme digestions (single digestion by E.coRI
and double by BgnI-XhoI) and specific PCR using PSHUTF/PSHUTR primerpairs (both binding on CMV promoter sequence, yielding a product of 2.6 kb) and chGMCSF-specific CHF/CHR primers (internal primers of chGMCSF, resulting in a product of 462 bp). Results indicated that the adenoviral DNA plasmid containing the chicken GMCSF gene (chGMCSF) was obtained, and that was confirmed as pAd-pShuttle-KZchGMCSF. Successful construction hereby described, provides vector materials for subsequent construction of the chGMCSF-recombinant adenovirus in the HEK293 cells in the next stage.