Immune-enhancing Effect Of S.typhimurium Flagellin On Bovine S.aureus GapC Protein

Staphylococcus aureus is one of the major pathogens cause of dairy cows mastitis,multdrug-resistant S. aureus infections are ever increasing and some strains respond to few, if any,conventional antibiotic therapies. So, vaccine development become the key to prevention of dairycows mastitis. Research shows that, the high conserved S. aureus surface antigen GapC, whichwith the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, has a goodimmunogenicity and immunoprotection. Flagellins as the ligands of Toll-like receptor5(TLR5),can induce innate immune responses. In particular, flagellin is a safe and non-toxic able tostimulate cellular immunity new immune adjuvant, which found in recent years. In this study, weuse E. coli expression system to express Flic protein of Salmonella typhimurium and the fusionprotein Flic-GapC, analyzed their immunogenicity and immunoprotection against S. aureusinfection, and discussed the adjuvant effec of flagellin, at the same time providing new method forthe cows mastitis research.First, flic gene of Salmonella typhimurium was amplified by PCR, cloning to pMD18-Tvector subsequently. The recombinant plasmid, pMD-T/flic, was sequenced. We then comparedand analyzed this sequence with the template sequence. Then, cloned fliC gene to pQE-30expressvector and translated into E.coli XL1-Blue strain. After IPTG induction, identified therecombinant protein by SDS-PAGE and western blot. Secondly, through the overlap polymerasechain reaction, amplified fliC-gapC fusion gene, cloned to pQE-30express vector and translatinginto E.coli XL1-Blue strain. After IPTG induction、SDS-PAGE and western blot detection,purified recombinant protein by nickel column, detected their GAPDH activity, and immunizedmice. Mice were divided into six groups and immunized with recombinant protein Flic、GapC,recombinant protein GapC emulsified with Freud’s adjuvant, fusion protein Flic-GapC, combinedprotein Flic+GapC and PBS respectively. Mice were immunized s.c. with recombinant protein,and received boost immunization three weeks after primary immunization, and were challengedwith S.aureus strains Wood46three weeks after boost immunization, and survival was monitoredfor one week. At the same time, Enzyme-Linked Immunosorbnent Assay (ELISA) was used tomeasure the levels of IgG antibody titres in mice serums. Two weeks after the secondaryimmunization, isolate spleen lymphocytes, then conduct lymphocyte proliferation experiment.Finally, the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by Elispot assay.This research has expressed the recombinant protein Flic and Flic-GapC in E.coli XL1-Bluesuccessfully.The group of GapC protein+Freud’s adjuvant, FliC-GapC protein, Flic protein+GapCprotein antibody growth is obvious, the IgG antibody titers in serum from immunized mice at14dafter boost immunization were highest, which were1:64000,1:64000and1:32000respectively.The spleen lymphocyte stimulation index as well as the cells secereting IFN-γ and IL-4increasedsignificantly compared with control group. A week after challenge, the three groups of immune protection rate higher than the other groups, the protection rate were80%,70%and60%,respectively, while a separate GapC protein protection only20%. These results indicate that, thefusion protein Flic-GapC can elicite better cellular immunity and humoral immunity, and itsprotection rate was higher than the group of Flic protein+GapC protein, but lower than the groupof GapC protein+Freud’s adjuvant.In summary, flagellin as an immune adjuvant, can significantly enhance the immunogenicityof the GapC protein, elicit mice producing GapC-specific IgG, and stimulate the cellular immuneresponse of the mice, showing a good immune adjuvant effect. which provided a theoretical basisfor the development of highly efficient vaccine against dairy cows mastitis. Meanwhile,Flic-GapC fusion protein also protect mice against S. aureus challenge to some extent, whichprovided a theoretical basis for the development of highly efficient vaccine against dairy cowsmastitis.