Analysis Of B Cell Epitopes Of Staphylococcus Aureus GapC

Staphylococcus aureus?S.aureus?,one of the most important pathogens,causes human epidermal infection,sepsis,pneumonia,infective endocarditis and septic arthritis,which poses a serious threat to human health.S.aureus can also cause animal infections,such as chicken skin pustules,acute and chronic mastitis in cattle and sheep,pig abortion and dermatitis,causing huge economic losses to animal husbandry.Due to the abuse of antibiotics,the resistance of S.aureus is increasing.So there is an urgent need to develop new immunotherapies and immunoprophylaxis.Previous studies have found that the GapC protein of Staphylococcus aureus is highly conserved,which is a surface protein with high GAPDH activity,transferrin binding activity and some other biological activities.This protein can induce an effective humoral immune response in vivo.However,the B cell epitopes of S.aureus GapC have not been well identified.In this study,primers were designed according to published S.aureus gapC gene sequence.The gapC gene was amplified by PCR and ligated with pET32a?+?plasmid to express the recombinant protein GapC.The purified rGapC protein was used as an immunogen and 6-8 weeks old female BALB/c mice were intramuscularly inoculated.Two hybridoma cell lines,1F4 and 2A9,secreting stable antibodies were screened by hybridoma technology and limited dilution method.We identified the subclass,specificity and the effect on GAPDH activity and performed a passive immune protection experiment.The mAbs were found to be of IgG1 type,binding specifically to S.aureus GapC protein,and were able to inhibit the GAPDH activity of rGapC.Mice were challenged after passive immunization with mAbs,and the survival time of the mice injected with mAbs before challenge was longer than that of the mice in the control group.We screened the motif ²³⁶PVATGSLT²4343 and ²⁷²GYTEDEIV²⁷⁹,which were recognized by m Ab2A9 and mAb1F4 through phage display system.Artificial synthesizing epitope genes were expressed in E.coli BL21.We analyzed the key amino acids of the epitopes by amino acid site-directed mutation test.P236 G240L242 T243 were formed the core of ²³⁶PVATGSLT²4343 and G272 D276 E277 I278V279 were formed the core of ²⁷²GYTEDEIV²⁷⁹.Indirect immunofluorescence assay was used to confirm that the epitopes were exposed on the surface of the cells.At the same time,the B-cell epitope antisera promoted the opsonophagocytic activity of macrophages.Furthermore,we showed that epitope-peptides could induce protective immune response against S.aureus infection in the immunized mice.Our results will be useful for the further study of epitope-based vaccines against S.aureus infection.