Preparation Of McAbs Against S.aureus TRAP And Analysis Of Eptitopes To The McAbs

Staphylococcus aureus, a gram-positive bacterium, is an important pathogen that can infect human andanimals. More and more drug-resistant S.aureus strains are emerging during antibiotic abusing, which makesthe treatment of S.aureus infection very difficult. So more research focus on prevention of S.aureus infectionrather than treatment. Some proteins, which can prevent and treat bacterial infection, become the candidate fordevelopment of vaccines. TRAP, a unique protein of staphylococcus, is an ideal one of them, whichimmunoprotection against S.aureus infection is very effective, but its cellular localization and functions arestill unclear. In this study, anti-TRAP monoclonal antibodies were prepared and screened and theircorresponding epitopes in TRAP ware respectively analysized. This is essential for development of epitopevaccine, cellular localization of TRAP and function analysis of TRAP.In order to prepare anti-TRAP McAbs, BALB/c mice were immunized with recombinant TRAP forhybridoma fusion, and11clones secreting anti-TRAP McAbs were obtained after clone screening, which werenamed as2A1,3A6,3B1,1B4,2C5,4C7,5C3,2D8,2A7,3A1, and1C4. Among them, the heavy chains of8clones were IgG1subclass, including2A1,3A6,3B1,1B4,2C5,4C7,5C3,2D8and that of3clones were IgMsubclass, including2A7,3A1, and1C4. The light chains of them all were κ sub-class. The hybridomasupernatant of the8clones secreting IgG1could specifically recognize the recombinant TRAP protein bywestern blot, and showed no cross-reaction to other proteins used in this research and had goodimmuno-reactivity with recombinant TRAP by ELISA.The8IgG1McAbs were used to biopan a12-mer phage peptide library three times. The selected phageclones were identified by ELISA and then sequenced. The amino acid sequences probed with the McAbs byphage display were analyzed, and3epitopes were identified. They are probably2linear epitopes and aconformational epitope, and the amino acid sequences of the3epitopes are respectively YLYP, SYFE andL-G-L. Representative McAbs recongnizing the3epitopes have strong opsonophagocytic activity, and the3epitopes are confirmed exposing on S. aureus surface. Further analysis of the epitopes is following.