Construction And Immunogenicity Of An E.Coli Strain Displaying The Lpp'OmpA-TRAP Fusion Protein On Surface

Bovine mastitis is one of the most prevalent diseases affecting the dairy industry worldwidely.It is a complex disease mainly caused by bacteria,such as S.aureus and E.coli.Abuse of antibiotics in the treatment has resulted in an increased risk of resistant strains emerging that may enter the food chain.So vaccine immunization was payed more attention.Among the vaccines against bovine mastitis,S.aureus inactivated whole-organism vaccines,capsular polysaccharide conjugate vaccines have been shown failure,while subunit vaccines have showed certain immunoprotection.But due to the weak cellular immune response and complicated proparation processes,subunit vaccines have not been put into production.So,the present study aims at to construct a recombinant E.coli strain displaying S.aureus TRAP on its surface,and to investigate its immunogenicity.This can provide the reference for the research on vaccines against dairy cow mastitis.In this experiment,the lpp'omp A and lpp'ompA-traP gene were fused by over-lap PCR and then ligated into expression plasmid pQE30 producing pLO and pLO-TRAP.These two recombinant plasmids were transformed into XL1-Blue,resulting in XL1-Blue/pLO and XL1-Blue/pLO-TRAP.The recombinant strains wer induced to express the fusion protein TRAP.The expressed ptoduct was correspond to a expected molecular weight.The results of whole cell ELISA,FACS analysis and LSCM observation confirmed that TRAP was displayed on the surface of XL1-Blue through the Lpp'OmpA surface display system.ICR mice were intramuscularly immunized with recombinant strains XL1-Blue/pLO,XL1-Blue/pLO-TRAP and recombinant protein TRAP.The blood of immune mice was collected to check the anti-TRAP antibody levels at different time.The lymphocytes secreted IL-4 and IFN-? were detected by ELISPOT and that secreted IL-17 A were detected by indirect ELISA.At last,immunized mice were challenged with S.aureas Newman and HLJ23-1.XL1-Blue/p LO-TRAP and TRAP protein immunized group all produced produce good cellular and humoral immunity.