| Staphylococcus aureus(S.aureus)is common of causative agent of bovine mastitis,The disease is long duration,disease that is difficult to treat and prevent,This disease has caused economic loss in dairy industry.Due to the serious drug resistance of S.aureus,immune control is an important measure to resist S.aureus infection.GapC(Glyceraldehyde-3-phosphate dehydrogenase)protein is Staphylococcus aureus surface protein has GAPDH activity,which has batter immunogenicity and regared as a good candidate antigen.Presently,there are no effective prevention and treatment measures for S.aureus dairy mastitis of cow,and the immune characteristics and combined immune effect of local isolates in Xinjiang are not clear.In this study,Staphylococcus aureus 15119 strain was isolated and identified,and GapC was amplified and expression recombinant p ET-28a-GapC-15119 was constructed,GapC protein with molecular weight of about 44 k Da were induced and purified.Western blot results showed the recombinant proteins had good immunogenicity.The purified GapC and ClfA,EsxB protein were used to immunize mice.The epitope of GapC was predicted using bioinformatics method epitope peptides were synthesized.The serum antibody level,titer,subtypes of immunized mice and antigenicity of epitope peptides was investigated by ELISA.The results showed that the antibody level and titer of the GapC+EsxB+ClfA immune group was higher than that of the other experimental groups,the highest antibody titer was 1:72900,the antibody subtype of all immune group mainly were Ig G1 and Ig G2 b.The antigenicity test showed that PC 9 and PC 10 conformational epitope peptides had better antigenicity,then followed by PL 7 and PL5 linear epitope polypeptides.The immunized mice were challenged with Staphylococcus aureus 15119,the immune effect of each group was analyzed through the organ load method,protection rate,body weight and histopathological observation.The bacteria load results indicated that GapC+EsxB+ClfA immunization group had less bacteria load than other immune groups.The results showed that the challenge protection rate of ClfA,GapC+ClfA and GapC+EsxB+ClfA immunization group was 100%,15119 and GapC+EsxB group was90.9%,EsxB group was 81.8%,EsxB group has the lowest of protection rate was 63.6%.And the weight records showed that GapC+EsxB+ClfA immunization group had the fastest weight recovery;The histopathological results of spleen and kidney showed that the GapC+EsxB+ClfA immunization group had the best protective effect.Using Salmonella equine producing and S.aureus vector and recombinant plasmids of ClfA,Fnbp A to immunize dairy cows.Then content of cryptic milk,milk fat,milk protein,lactose,SCC index and antibody in immune cows were tested.The results showed that the recombinant plasmids of ClfA,Fnbp A had good immune effect on dairy cows,S-ClfA+Fnbp A(200 ?g)immune group had the highest antibody level in bovine serum,antibody titers can reach 1:24 300;the S-ClfA+Fnbp A immunization stimulate higher levels of IL-4 and IFN-? in the body and indicating.To stimulate Th1 and Th2 immune response were stimulated;The hermitage rate and somatic cell results showed that S-ClfA+Fnbp A immune group could effectively reduce the incidence of mastitis in dairy cows,it also could raised the content of milk fat,milk protein and lactose.In summary,the combination of GapC,EsxB and ClfA protein of S.aureus has good immune effect on mice,and the predicted epitope peptide of GapC protein has good antigenicity,which provides a certain basis for the subsequent animal immunity,and the dairy cow immune experiment show that the effectiveness of the immunization with the p VAX1-ClfA,p VAX1-Fnbp A recombinant plasmid based on the bacterial vector and this result also provide an experimental basis for the later stage of GapC recombinant plasmid construction and animal immunity,also provide a reference for the prevention of dairy cow mastitis. |
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