Immunogenicity Of Streptococcus GapC_1-150aa Displayed On The Surface Of E. Coli XL1-blue Strain

Bovine mastitis is one of the most prevalent diseases affecting the dairy industry world widely.It is a complex disease mainly caused by bacteria, such as S.agalactiae, S.dysagalactiae, S.uberis,S. aureus and E. coli. Abuse of antibiotics in the treatment has resulted in an increasing risk ofresistant strains emerging and antibiotic-containing milk antibiotic-containing milk wasforbidden on food market. So vaccination against bovine mastitis has been paid more attention.GapC protein is one of virulent factors expressed on the surface of Streptococci, which has goodantigenicity and cross immune protection in the three Streptococcus species. Our previous datahave shown that S.dysgalactiae GapC1-150aaprotein had the similar immunoprotection comparedwith the full-length GapC. So, the present study aims to construct a recombinant E.coli XL1-bluestrain displaying Streptococcus GapC1-150aaon its surface, and to investigate the immunogenicityof the GapC1-150aaon the E.coli surface.In this experiment, the ompA gene of the E.coli XL1-blue strain was replaced withlpp’ompA-gapC1-cm gene or lpp’ompA-cm by Red recombinantion. In this way, GapC1-150aacould be expressed on E.coli surface as a fusion protein with1~159aa of OmpA. Primers weredesigned according to the reported gene sequences encoding gapC1, ompA and cm on pKD3.lpp’ompA-gapC1-150aaor lpp’ompA genes were ligated with cm by overlap PCR. The twofragments were respectively electroporated into the E.coli XL1-blue strain containing pKD46plasmid. The recombinant strains, named as XL1-Blue/LOG76and XL1-Blue/LO, were inducedto express the fusion protein GapC1-150aaon the surface. The molecular weight of the expressedfusion protein by XL1-Blue/LOG76corresponded to the expected molecular weight. TheGapC1-150aawas confirmed displaying on the surface of XL1-Blue by indirect ELISA with platecoated with the recombinant E.coli strain, FACS analysis and LSCM observation.Then, ICR mice were intramuscularly immunized with the recombinant strains XL1-Blue/LO,XL1-Blue/LOG76or the recombinant protein GapC1-150aa. The sera of the immunized mice werecollected and the anti-GapC1-150aaantibody levels in the sera were detected by ELISA. Thelymphocytes secreting IL-4and IFN-γ were detected by ELISPOT and IL-17A level in thesupernatant of cultured spleenic lymphocytes was detected by indirect ELISA. At last, theimmunized mice were challenged with S.uberis SD0306, S.dysgalactiae LS0312andS.agalactiae LS0310. The mice immunized with XL1-Blue/LOG76or GapC1-150aaproteinproduced better cellular and humoral immunity. The results above indicate that it is feasible todisplay GapC1-150aaantigen on E.coli surface as a vaccine against streptococcus infections.