The Perliminary Study On The HPV16E7Antigen-restricted CTL Epitope Of The Tu Nationality Women From Wufeng Country Of Hubei Province

OBJECTIVE:Perliminary study on the HPV16E7antigen-restricted CTL epitope in the Tunationality from Wufeng country of Hubei province in china,it can lay the foundationfor the further research of the epitope candidates’ antigenicity identification andclinical experiment.METHODS:To predict the CTL epitopes of E7antigen by NetMHCpan software,the peptideswere synthesized with solid phase strategies,purified with reverse-phase HPLC andidentified with mass spectrometry;Respectively select30healthy women and primarycervical cancers of the Tu nationality from Wufeng country of Hubei province,DNAextracted from peripheral blood and HLA genotyping was performed on aMulti-Analyte Profiling system (xMAP)(Luminex HLA-SSO) using a WAKFlowHLA typing kit, We used E7peptide stimulate with peripheral blood lymphocytesrespectively obtained from five HLA-A*02+heathy women and HLA-B*07+cervicalcancers in vitro for immunology experiment,We then detected the number of theINF-gamma spots（Spots Forming Cells,SFCs）and analyzed the difference in theHPV16E7-specific-T-cell immune responses using the statistical methods.RESULTS:The purity of the peptides were＞95%and the measured values of theirmolecular weight were identical with theoretical valus,which were identified as highpurity of target peptides.Experimental results indicated that the E7eptides induced strong CTLs immuneresponses and secreted high level of INF-gamma spots in HLA-A*02+healthygroups.Through statistical analysis,it was found that there were significant differencesbetween the groups after analysis of variance（F=15.17,P＜0.001）,we also found the statistically significant differences between the experimental gruops and negativecontrol group after LSD test,P2:E741-55vs P4:E751-65were most significantly(P<0.05),no significant difference between the two.Experimental results indicated that the E7eptides induced weak CTLs immuneresponses and secreted low level of INF-gamma spots in HLA-B*07+cervical cancergroups.Through statistical analysis,it was found that there were significant differencesbetween the groups after analysis of variance（F=40.26,P＜0.001）,we also found thestatistically significant differences between the experimental groups and negativecontrol group after LSD test,P5:E766-80was most significantly (P<0.05).Experimental results indicated that the level of INF-gamma spots between theHLA-A*02+healthy groups and HLA-B*07+cervical cancer groups were significantlydifferent, and the difference was statistically significant (F=268.82, P <0.001).Through SNK pairwise comparison, we found that P2: E741-55,P4: E751-65and P5:E766-80had the most significant impact on the results and the difference wasstatistically significant compared with other polypeptides(P <0.05), but among thethree is not yet considered statistically significant.CONCLUSIONS：HLA-A*02can effectively present the HPV16E7antigen peptides to inducesignificant CTLs immune response;HLA-B*07can’t effectively present the HPV16E7antigen peptides and inducesignificant CTLs immune response.E741-55(PAGQAEPDRAHYNIV) and E751-65(HYNIVTFCCKCDSTL) have antigenicity,andmay be the CTLs epitope peptide candidates for the subsequent study.