Preparation Of McAbs Against Streptococcus Dysgalactiae GapC_1-150aa And Analysis Of B Cell Epitopes To The McAbs

Bovine mastitis, caused by various pathogens, leads to serious economic losses to the dairy industry.Streptococci are the most frequently pathogens causing the mastitis, which incudes Streptococcusdysgalactiae, Streptococcus agalactiae and Streptococcus uberis. GapC protein is one of virulence factorsexpressed on the surface of Streptococci, which has good antigenicity and cross immune protection in thethree Streptococcus subtypes. Previous dates have shown that S. dysgalactiae GapC_1-150aaprotein had thesimilar immune protection compared with the full-length GapC in our lab. Therefore, in order to understandthe protective mechanism of GapC from Streptococcus, monoclonal antibodies against GapC_1-150aawereprepared and epitopes to the McAbs were analyzed using phage display technology.4-6week-old female BALB/c mice were immunized with purified recombinant GapC_1-150aaprotein, andhybridomas secreted11strains McAbs were obtained by using hybridomas screening. McAbs from ascitesfluids of BALB/c mice were purified with rProteinG kits. The specificity and sensitivity of McAbs weredetermined by isotypes identification, crossover trial, stability test and affinity test. The specificity ofmonoclonal antibodies against recombinant GapC_1-150aawas detected by Western blotting.The epitopes to the McAbs were screened using phage display technology and the positive phage cloneswere detected by the indirect ELISA. The high-affinity positive clones were sequenced and the sequenceswere analyzed by the bioinformatic software. The representative McAb1F2,1E11,5B7held the three motifand their corresponding amino acid sequence were TRINDLT、TGFFASK、DTTQGRFDGT, respectively.Alanine was used to substitute the amino acid one by one in each epitope and the site-mutated epitopes wereexpressed in E. coli. The key amino acids of each epitope were identified through western blotting using thecorresponding monoclonal antibody. The three mAbs had very significant opsonization in opsonophagocytictests. The three epitopes were identified exposing on the surface of bacteria by ELISA coating with the wholebacterial cell. Sequences of the three epitopes were highly conserved in all Streptococci recorded in GenBank.Mice were intravenously immunized with McAbs against the three epitopes and stayed alive at24h after thechallenge of S. dysgalactiae compared with the control group. These demonstrate that these epitopes play keyroles in inducing immune protection.