| Mastitis is the most common and costly disease in dairy cow industry.Previous studies indicated that mastitis is an inflammatory response of breast tissue caused by invasion of pathogens.And the main pathogens of mastitis include Staphylococcus aureus,Streptococcus agalactiae,coliform.However,the increasing of mastitis pathogen resistance and drug residue caused by antibiotic misuse affected the treatment effect of antibiotic.Therefore,taking precautions against Dairy Mastitis with vaccine has become a hot research topic.This study identified Staphylococcus aureus(J581)and Streptococcus agalactiae(A20)isolated from mastitis cows as vaccine strains.In this study,we extracted capsular polysaccharide(CP)from J581 and A20 then purified it,furthermore conjugated with tetanus toxoid(TT).Finally,studied the immune effect of conjugate vaccine preliminarily.The separation and purification process of CP for Staphylococcus aureus and Streptococcus agalactiae were established.Using the modified enzyme method to extract capsule polysaccharide from the precipitation of Staphylococcus aureus and Streptococcus agalactiae.The crude polysaccharide was treated by Sevage method to remove proteins in CP,then over 10 KDa and30KDa for two times ultrafiltration.The ultrafiltrate was purified by CL-4B agarose gel column,and purified CP was obtained.The results showed that the final yield of Staphylococcus aureus CP was 49.85 mg/L,the content of protein was 0.923%,0.867% for nucleic acid.And the final yield of Streptococcus agalactiae CP was 57.05 mg/L,the content of protein was 0.917%,0.887% for nucleic acid.Purified CP was derivatized with adipic dihydrazide(ADH).The derivation was then conjugated with TT under the action of ethyl-dimethyl-amino-propyl carbodiimide(EDAC).Multi-wave length scanning was used to identify the conjugate materials.Compared with the maximum absorption peak of poly saccharide,the absorption peak of the conjugate was shifted,which indicated that the coupling of multiple glycoproteins was successful.When the CP and TT were coupled at the mass ratio of 1:1,the coupling effect was the best.The coupling rate ofStaphylococcus aureus conjugate was 1.00,and the coupling rate of Streptococcus agalactiae conjugate was 0.967.The CP-TT conjugated vaccine were diluted by PBS.6 kinds of CP-TT conjugated vaccine of which content of polysaccharide were respectively 5 ?g/m L,10 ?g/m L,15 ?g/m L prepared from Staphylococcus aureus and Streptococcus agalactiae.The mixed CP-TT conjugated vaccine content polysaccharide 10 ?g/m L prepared from Staphylococcus aureus and Streptococcus agalactiae.210 Kunming female mice were randomly divided into 9 groups,including 8 experimental groups and 1 control group.The immunization of 7 kinds of CP-TT conjugated vaccine and mastitis inactivated vaccine was carried out inmice.Thenmeasured the antibody determination and Ig G level of mice at different time periods after immunization.Finally,the effect of anti artificial infection was carried out at 35 d after immunization.The results showed that the antibody level and the protection rate of the CP-TT conjugated vaccine with polysaccharide content of 10 ?g/m L were the highest.The effect of CP-TT conjugated vaccine of Streptococcus agalactiae was much better than CP-TT conjugated vaccine of Staphylococcus aureus.In conclusion,the antibody level and the protection rate of mixed CP-TT conjugate vaccine > CP-TT conjugate vaccine of Streptococcus agalactiae>CP-TT conjugate vaccine of Staphylococcus aureus.This research established the separation and purification process of CP for Staphylococcus aureus and Streptococcus agalactiae,and prepared the CP-TT conjugate vaccine successful.Through this study the optimal polysaccharide content in conjugate vaccine was determined.Besides,our results provide a proof of further research onmastitis vaccine and improve the immune prevention effect of dairy cow mastitis. |
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