Immunogenicity And Immunoprotection Of An E. Coli Strain Displaying S. Aureus TRAP On Its Surface

Mastitis is one of the most common diseases of dairy cows. Staphylococcus aureus(S.aureus) and Escherichia coli(E.coli) are of higher rates among the pathogens causing mastitis.In order to develop a vaccine against both S. aureus and E. coli, the conserved TRAP of S.aureus was displayed on the surface of E. coli strain BL21(DE3) by fusing TRAP to the outer membrane C(OmpC) of the E. coli. The immunogenicity and immunoprotection of the strain were then evaluated.In this study, S. aureus TRAP gene and E.coli BL21(DE3) OmpC gene were fused by overlap PCR, and OmpC-TRAP gene and OmpC gene were respectively inserted into the plasmid pET-28 a. The recombinant plasmids were named pCT and pC. These two recombinant plasmids were transformed into BL21(DE3), and the positive expression strains were named BL21/pCT and BL21/pC. The recombinant protein, expressed by the strain BL21/pCT or BL21/pC after IPTG induction, has the molecular weight as predicted, and was confirmed to be displayed on the surface of E. coli BL21(DE3) by whole cell Western-blot, ELISA and LSCM.Eight-week old ICR mice were divided into 4 groups as follows, BL21/pCT group,BL21/pC group, TRAP group, and PBS group, namely. During the primary immunization, each mouse was intramuscularly immunized with 2×108 cfu BL21/pCT, BL21/pC, or 100 μg TRAP emulsified with Freund’s complete adjuvant. A booster immunization was performed 3 weeks later with antigens in Freund’s incomplete adjuvant emulsion. After the challenge with HLJ23-1,Newman, or 2002-1, the survival rates of mice in PBS group were 10%, 0%, and 0%,respectively. And that in BL21/pCT group was 70%, 70%, and 40%. One week after the booster immunization, specific cytokines secreted by splenic lymphocytes were detected using Elispot or ELISA. Two weeks after the booster immunization, antibody titers, antibody subclasses, and the neutralizing capacity of antibody in serum were detected with indirect-ELISA. Meanwhile, the mice were challenged with S. aureus strain HLJ23-1, Newman or E. coli 2002-1, respectively.Results showed that high antibody level in the sera of BL21/pCT-immunized mice was detected,compared to that of other immunized mice, IgG1 and IgG2 a were the main antibody subclasses,and high levels of IFN-γ and IL-4 were detected. These data indicated that the BL21/pCT group exhibited better cellular and humoral immunity. These results suggested that BL21/pCT, aconstructed E. coli strain displaying the S. aureus protein TRAP on its surface, protected the mice against both S. aureus and E. coli infections. This research will provide a reference for the development of novel vaccines against the bovine mastitis caused by S. aureus and E. coli.