Field Trial Of Triple Inactivated Vaccine Against Mastitis Of Cows And Cloning And Expression CAMP Gene Of Streptococcus Agalactiae From Mastitis Milk

Appropriate adjunvant was selected to make triple inactivated vaccine against mastitis of dairy cows and the new vaccine was tested with stability, sterility and safety. The results showed the vaccine against mastitis of cows emulsified with oil adjuvant was steady, germfree and safe. In the mouse test model, serology test and challenge test were processed after three times immunization with the new vaccine. The results showed after three times inoculation with the new vaccine high specific antibody titers could be elicited and effective protection against homogenate and heterogenate S. aureus , E.coli, S.agalactiae could be gained.To evaluate the effect of the new inactivated vaccine, field trial was carried on. The whole bacterial cells of S. aureus , E.coli, S.agalactiae antigen-based indirect ELISA was established to detect the antibody titers of sera of dairy cows. After 3 times vaccination, the antibody titers were evaluated, and the results indicated that the antibody titers of sera of tested cows were dramatically higher than those of controlled cows respectively. From the bactericidal activity of milk whey in vitro, it could be concluded that in the milk whey the higher specific antibody titer is attained, the higher bactericidal activity is expected.CAMP factor gene was cloned from genome DNA of Streptococcus agalactiae by PCR, and the PCR product was inserted into vector pMD18-T. Sequence analysis showed that the Streptococcus agalactiae CAMP gene(cfb) was composed of 768 nucleotides encoding a mature polypeptide 255 amino acids. Compared to other Streptococcus agalactiae CAMP gene (cfb) that reported shared the identity of 98.4%. The positive colony which were identified by digestion and PCR showed that the recombinant plasmid pET32a+/ cfb was constructed successfully. SDS-PAGE result showed that recombinant protein, with a molecular weight of 48 KD, was expressed in inclusion bodies in E.coli and the recombinant protein accounted for 52.7% of the total proteins.This study provides conditions for the research of pathogenic and immune mechanism and vaccine design.