In order to study the immunogenic and protective effects of Sip-GAPDH from Streptococcus agalactiae recombined chimeric DNA vaccine for Oreochromis niloticus,we cloned Sip and GAPDH gene by PCR,and then obtained the Sip-GAPDH fusion gene using the splicing overlap extension technology(SOEing).The Sip-GAPDH fusion gene was inserted into pcDNA3.1(+)vector and confirmed by restriction endonuclease digestion,PCR amplification and sequencing,and O.niloticus were immunized with the recombined plasmid(designated as pcDNA-Sip-GAPDH)by intramuscular injection.The pcDNA-Sip-GAPDH expression in tissues(including muscle,brain,head kidney,spleen,gill and liver)was analyzed by PCR and RT-PCR at 7 and 28 d after immunization.To reveal the serum antibody titer,expression of immunogenes(IgM,IL-1β and CD8)and relative percentage survival(RPS),we used the enzyme-linked immunosorbent assay(ELISA),real-time PCR and bacterial challenge in vitro to demonstrate the DNA vaccine is a potential candidate for vaccine development of O.niloticus. The results indicated that pcDNA-Sip expression could be found in all studied tissues and antibody titer rose to peak(1:4 096)at 21 d after immunization.Quantitative real-time PCR(qRT-PCR)analysis showed that IgM,IL-1β and CD8 had relatively high expression levels in the head kidney,thymus,spleen.After S.agalactiae infection,transcripts of IgM,IL-1β and CD8 increased and reached its peak 12 h in thymus and head kidney,and 48 h in spleen,respectively.Additionally,the relative percentage survival(RPS)value was 93.3% according to the mortality by S.agalactiae challenge.All the results suggested that the chimeric DNA vaccine is an effective vaccine candidate against S.agalactiae infection.