| Therapeutic peptide vaccine of carcinoma is one of the research highlights in targeted immune therapy,it approaches to active immunization induce specific anti-tumor effect by cytotoxic T lymphocytes.Preliminary studies of our research group selected antigen TM4SF1 from the c DNA constructed using cancer cells from ascites of patients with epithelial ovarian cancer,and found that TM4SF1 expressed higher in epithelial ovarian cancer tissues than benign ovarian tumor tissues and normal ovarian tissues.And it is closely related to the development of ovarian cancer.Dates of research indicate that TM4SF1 is a potential target for immunotherapy on ovarian tumor.Aims of This research are to predict potential CTL epitopes restricted by HLA-A2 molecule of TM4SF1,to assay the affinity and binding stability properties of potential CTL epitopes with HLA-A2 molecule.Epitopes with better stablilty are jointed with adjuvant and immunized to Tg(HLA-A2.1)1Enge/J mice.CTL epitopes restricted by HLA-A2 molecule of TM4SF1 with immunological competence in vivo are screened and identified through assaying peptide-specific T cell responses by ELISPOT.Part1.Prediction of HLA-A2 Restricted CTL Epitopes from Ovarian cancer associated antigen TM4SF1 Objective To predict CTL epitopes restricted by HLA-A2 molecule of TM4SF1 through combining a variety of forecasting software.Methods CTL epitopes restricted by HLA-A2 molecule of TM4SF1 are predicted by BIMAS,SYFPEITHI,IEDB,PROPREDⅠaccording the amino acid sequence of TM4SF1 searched from Pubmed detebase.Epitopes are ranked according the score through comprehensive analysis of the predicting outcomes from prediction softwares above.Results The top ten candidate CTL epitopes restricted by HLA-A2 molecule of TM4SF1 are screened from 17 candidate epitopes according score through comprehensive analysis of the predicting outcomes from prediction softwares above and named P1 to P10.Conclusions Combined four different forecasting software is a effective strategy to get better candidate epitopes and avoid limitations of a single software.Part2.Affinity detection of HLA-A2 Restricted CTL Epitope from Ovarian cancer associated antigen TM4SF1Objective To detecting the affinity and binding stability of candidate HLA-A2 Restricted CTL Epitopes of TM4SF1 from Part1 study with HLA-A2 molecular.Methods 10 candidate peptides,one positive control peptide and one negative control peptide are synthesised by commercial company.A reference system is establish.T2 cells were incubated with low concentrations of peptides and assay by flow cytometry to get the mean intensity of fluorescence(MIF)of HLA-A2 molecular on T cells surface.Using flourescence index to measured The affinity of peptides with HLA-A2 molecules are divided into three levels(FI>1.5: high affinity,1.0<FI <1.5: moderate affinity,FI<1.0: low affinity)according fluorescence index.Binding stability of high affinity and low affinity peptides with HLA-A2 molecular are assay at conditions that T2 cells are incubated with different peptides concentration(10μ g/ml,20μ g/ml,30μ g/ml,40μ g/ml),and explore the relation of concentrations and MIF.Result In the referring system,the MIF of negative polypeptides group has no difference with the blank staining group:(6.60±0.95)vs(6.95±0.05),P>0.05.And the MIF of positive polypeptides was significantly higher than the blank staining group,(15.97±1.18)vs(6.95± 0.05),there is a statistically significant,P <0.05.Dates prove reference system is reliabe.Epitope P1,P2,P3,P6,P7 have a high affinity with HLA-A2 molecules(FI>1.5),polypeptide P4 has moderate affinity(1.0<IF<1.5),and P5,P8,P9,P10 have lower affinity(IF <1.0).Two highest affinity polypeptides(P1,P2)and two low affinityspeptides(P8,P10)are selected to assay binding stability.Datas show that MIF increases with increasing concentrations of peptide,P <0.05,the regression coefficients of four peptides were 0.82,0.59,0.19,0.35,suggesting that the candidate polypeptide P1,P2,P8,P10 all have binding stability.Conclusion Results of affinity test in vitro are reliable,Epitope P1,P2,P3,P6,P7 have a high affinity,P4 has the moderate affinity,and epitope P5,P8,P9,P10 have a low affinity.polypeptides P1,P2,P8,P10 have the binding stability and can be used for further research.Part3.Immunoreactivity Assay in vivo of HLA-A2 Restricted CTL Epitope from Ovarian cancer associated antigen TM4SF1Objective To assay the immunoreactivity of candidate CTL epitopes peptide(P1、P2、P8、P10)in Tg(HLA-A2.1)1Enge/J mice.Methods Tg(HLA-A2.1)1Enge/J mice are immunized withcandidate epitope peptides(P1,P2,P8,P10)and adjuvant.Spleen lymphocytes are isolated from the mice and are used for direct ELISPOT tests and pre-culture ELISPOT tests.Through spots forming cells(SFC)number produced by positive control stimulation of PMA to judge the quality control of ELISPOT.The ability of activate CTL and the IFN-γ levels secreted by single activate cells are analyzed according the number of SFC’ net T(T=SFC number in experimental wells-SFC number in negative wells)and the average spot size.Comparing the results of direct ELISPOT with the pre-culture ELISPOT,and choosing a more sensitive text result as standard for analysis.Peptides with immunological activity are identified by the result above.Result PMA stimulation frequency of all groups is 103-104/105 PBMC cells,all the results of each group meet quality control standards.There is no SFC in negative control group,adjuvant control group and epitope peptide P2,P8.There aer spots in positive control peptide group,epitope peptide P1 and P10 groups.SFC of pre-cultured ELISPOT is significantly higher than direct ELISPOT : [Positive control peptide:(322±8)vs(169±22),P<0.05],[P1:(114±10)vs(37±7),P <0.05],[P10:(156±31)vs(52±8),P<0.05].And the average spot size(unit: 1E-3sq.mm)of pre-culture ELISPOT is significantly greater than the direct ELISPOT: [positive control peptide:(21.91±2.45)vs(13.8±1.76),P<0.05],[polypeptide P1:(12.9±088)vs(8.31±140),P<0.05],[polypeptide P10:(17.5± 3.85)vs(11.96±0.61),P<0.05].Dates show pre-culture method is more sensitive than direct ELISPOT.Results of pe-culture ELISPOT are selected to analysis: SCF T value of epitope peptide P10 is higher than P1: [P10(156±31)vs P1(114±10),P<0.05].There is no statistically significant between two peptides average spot size: [P10(17.5±3.85)P1 vs(12.9±0.88),P>0.05].Conclusion Pre-cultured ELISPOT can improve the sensitivity of ELISPOT in vitro.Two CTL epitope peptide P1 and P10 have immune activity.Epitope peptide P10 with lower affinity has a higher immune activity than P1 with higher affinity. |
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