HPV16E7CTL Mimotope Peptide And The Experiment Of Preliminary Immunological Effects

Methods:1According to the structure of the wild epitope peptide and the preliminary experimentalresult in human serum, it was determined which part of should be reconstruct, and usingmolecular dynamics simulations and calculation the binding free energy.2. The wild and mimetic CTL epitope peptides were synthesized with solid phase strategies,purified with reverse-phase HPLC and identified with mass spectrometry.3The analysis on the binding of the wild and mimetic CTL epitope peptides withHLA-A2molecules based on the feature of T2cells.4The LDH assay was carried out to study the killing activity of the wild and wild andmimetic CTL epitope peptides on target T2cells.Results：1According to the structure of wild epitope and preliminary experimental results,1-bitand4-bit was replaced by D-type acid substitution respectively, and we got three peptides:①(d) RAHYNIVTF,②RAHY (d) NIVTF,③(d) RAH (d) YNIVTF.2Molecular Dynamics results indicated that wild-type and epitope peptide bind relativelystable with MHC-I molecules, the connection of amino acids in the binding pocket wasalso reasonable.3The wild and mimetic epitope were synthesized on a solid-phase simultaneous multiplepeptide synthesizer based on the Fmoc strategy, and purified by C18reverse-phase HPLCto a purity of>95%.4The wild epitope had medium affinity, and①（d）RAHYNIVTF acids were highestaffinity with HLA-A2the strongest affinity, while the remaining was few affinity.5A standard LDH assay was arried out to study the killing activity of these CTLs on T2target cells. The wild-type was57.87%, and①epitope peptide effect was33.46%, theremaining showed no obvious effectConclusions：1The wild-epitope peptide was detected the vitro half-life in human serum in stability inthe time2The D-amino could improve the tolerance of enzymes in the in human serum ieffectively3The vitro experiments demonstrated that①(d) RAHYNIVTF can induce CTL, but it notas good as wild-type epitope peptide These all finding show althought①(d) RAHYNIVTF has lower than wild,it show morestability and higher affinity with HLA-A2. If we can improve it immune effect and it willprovide more evidence for the study of therapeutic vaccines...