卵巢癌相关抗原TM4SF1 HLA-A2限制性CTL表位预测及鉴定

目的 预测并筛选免疫反应性较强的四跨膜蛋白超家族成员1（transmembrane 4 L6 family member 1，TM4SF1） 人类白细胞抗原A2（human leukocyte antigen A2，HLA-A2）限制性T淋巴细胞（cytotoxic lymphocyte，CTL）表位肽。方法 联合4种软件（BIMAS、SYFPEITHI、IEDB、PROPRED I）对TM4SF1的 HLA-A2限制性CTL表位进行预测，并采用预培养法酶联免疫斑点法（enzyme-linked immunospot assy，ELISOPT）、直接法ELISOPT对所测得表位肽的免疫反应性进行鉴定。结果 4种不同软件共预测出10条表位多肽，对其中4条（P1、P2、P8、P10）进行免疫反应性验证。多肽活化CTL能力结果显示，预培养法ELISPOT产生斑点形成细胞（spvt forming cell，SFC）的净值T明显高于直接法ELISPOT：［（阳性对照肽：322±8 vs 169±22，P<0.05）、（多肽P1：114±10 vs 39±7，P<0.05）、（多肽 P10：156±31 vs 52±8，P<0.05）］。单个活化CTL细胞分泌INF-γ因子的水平检测结果显示，预培养法 ELISPOT 产生斑点的平均大小明显大于直接法 ELISPOT［（阳性对照肽：21.91±2.45 vs 13.80±1.76，P<0.05）、（多肽P1：12.90±0.88  vs 8.31±1.40，P<0.05）、（多肽P10：17.50±3.85 vs 11.96±0.61，P<0.05）］。表位多肽P1、P10均具有免疫原性，P10的免疫活性更高。结论 预培养法ELISPOT检测多肽的免疫反应性较直接法ELISPOT灵敏性更高，多种软件联合ELISPOT预培养法可有效筛选出免疫反应性更强的卵巢癌相关抗原TM4SF1 HLA-A2限制性CTL优势表位，其中P10的免疫活性最高。

Prediction and identification of HLA-A2-restricted CTL epitopes from ovarian cancer-associated antigen TM4SF1

 Objective To predict and identify HLA-A2-restricted CTL epitopes from ovarian cancer-associated antigen TM4SF1. Methods Four prediction programs（BIMAS，SYFPEITHI，IEDB，PROPRED I）were used to predict HLA-A2-restricted CTL epitopes of TM4SF1. Immunoreactivity of these predicted epitopes was measured using ELISOPT directly and in culture. Results Ten candidate CTL epitopes （P1-P10） were screened，and four （P1，P2，P8，P10） were analyzed further for immunoreactivity. SFC was significantly higher in culture ELISPOT than direct ELISPOT for the positive control peptide （322±8 vs 169±22，P<0.05），epitope P1 （114±10 vs 39±7，P<0.05），and epitope P10 （156±31 vs 52±8，P<0.05）. Similarly，average spot size was significantly greater in culture ELISPOT than direct ELISPOT for the positive control peptide （21.91±2.45 vs 13.80±1.76，P<0.05），P1 （12.90±0.88 vs 8.31±1.40，P<0.05），and P10 （17.50±3.85 vs 11.96±0.61，P<0.05）. These results show that culture ELISPOT is more sensitive than direct ELISPOT，and that the SFC T value is higher for epitope P10 than P1. Conclusions Combining four prediction programs is an effective strategy to identify better candidate epitopes and avoid limitations of a single prediction program. Culture ELISPOT is more sensitive than the direct assay for verifying the immunoreactivity of predicted epitopes. The immunoreactivity of P10 is strongest.