| In the south of China,tilapia is the main freshwater aquaculture species.In recent years,the tilapia industry caused a great economic losses because of the Streptococcus agalactiae disease.Till now,there are still have no effective medicines for this dieeases,so the development of vaccines can be an hopeful ways to prevent and control the infections.Nowadys,more and more researchers pays more attintion on subunit vaccines.In this study,based on the reviews of Streptococcus agalactiae surface proteins,we cloned and expressed the protein ?-enolase,phosphoglycerate kinas(PGK)and Glyceraldehyde 3-phosphate dehydrogenase(GAPDH),make it to the subunit vaccines respectively.1.According to BLAST homologous alignment and PCR,we get the ORF of ?-enolase,PGK,GAPDH gene.The ?-enolase's ORF is 1308 bp,435aa,49.4kDa;the PGK's ORF is 1046 bp,349aa,42 kDa and the PGK's ORF is 1011 bp,336aa,42 kDa.According to the Both of the three proteins lack signal peptide but the ?-enolase may form a transmembrane domain.2.Using prokaryotic expression and affinity chromatography technology,purified the expressed recombinant proteins ?-enolase,PGK,GAPDH respectively.3.Inactivated whole-cell vaccine,?-enolase ?PGK and GAPDH protein vaccines were prepared;tilapia was twice immunized by intraperitoneal injection,and challenged by S.agalactiae THN0901 strain at two weeks interval.The result showed that the RPS of ?-enolase group?PGK group and GAPDH group with PBS control were 31.70%,28.70%,48.12% respectively.The result also showed that the RPS of ?-enolase?PGK and GAPDH protein with S.agalactiae inactivated whole-cell vaccines groups with PBS control were 86.23%,68.67%,71.35% respectively.ELISA analysis showed that serum antibody titers appeared to increase in different degrees.The results of this study show that ?-enolase,PGK and GAPDH proteins have certain immunogenicity and can be used as a candidate gene of subunit vaccine.These proteins also can promte the immune protection effect of whole inactivated vaccine. |
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