Fermentation Technology Of Expressed Streptococcu Dysgalactiae,Staphylococcus Aureus Gap C Protein Recombination Engineering Bacteria And Immune Study In Experiment

In order to determine the expressed Streptococcu dysgalactiae/Gap C (TR/Gap C) and Staphylococcus aureus/Gap C (JP/Gap C) protein engineering bacterium of fermentation cultivation technique and recombinant protein subunits vaccine immune effect, this research project of the two strains at first were cultivated in the small training experiment, and optimized fermentation cultivation technique. Though the single factor experiment to decide TR/Gap C and JP/Gap C engineering bacteria optimal growth condition.The results showed temperature 37℃,pH7.0,medium volume 20%,inoculum age 12h,rotation speed 200rpm,and inoculum amount 1%,3% respectively. Engineering bacteria were induced with IPTG to express in using optimal growth condition in 5L fermentation tank, and accredited their activity and the content of expressed Gap C protein. The content of expressed TR/Gap C and JP/Gap C protein were 2.72mg/ml,2.23 mg/ml respectively, GAPDH active detection and Western Blot test showed that recombinant protein has higher GAPDH activity and antigenicity.Mice was immunized with a single and mixed recombinant Gap/C protein subunits oil emulsion vaccine respectively, though ELISA method and MTT test to detect serum IgG antibody level and the cellular immune effect.Mice in protein group and contrast group were challenged with S.agalactiae,S.dysgalactiae,S.uberis,S.aureus at the tenth day after strengthen immunization, The ELISA results showed that the IgG antibody titres against the JP/Gap C and mixing Gap C protein immune group in mice serums reached its peak at the second week after boost immunization. MTT assay results showed compared with the control group the difference was significant. The protection rate of mixing Gap C protein group was 70% via challenging by S.agalactiae strain, 80% via challenging by S.dysgalactiae,80% for S.uberis challenging,70% for S.aureus challenging.. The protection rate of JP/Gap C protein group was 70% via challenging by S.aureus.20 healthy lactating cows were selected, then they were divided into/TR Gap C,JP/Gap C,mixed Gap C protein and contrast test at random, they were injectted protein subunit vaccine in neck after two weeks, in contrast to pre immune.all the number of quarters which is infected bacteria and the number of mean SCC(somatic cell count) drop out after the 70d immune, but the contrast is opposite, the number of mean SCC is upgrade, ELISA results showed that the Ig G antibody titres against three Gap C in mice serums reached its peak at the third week after boost immunization.To sum up, the study shows that the expressed Gap C protein engineering bacteria by fermentation can be used in large project preparation subunit vaccine. Gap C subunit vaccine can stimulate specific body immunological reaction and reduce the number of bacteria and somatic cell count in milk mammary gland, improve the quality in milk, the research results laid a theoretical foundation for developing cow mastitis vaccine...