小鼠肝素酶H-2K^b限制性CTL表位预测及其MHC-I亲和力分析  

Prediction of H-2Kb restricted CTL epitope of heparanase and affinity assay of these epitopes with MHC-I molecule

作　　者：汤旭东[1] 万瑛[2] 陈婷[1] 熊震[1] 陈陵[1] 余松涛[1] 罗元辉[1] 杨仕明[1] 

机构地区：[1]第三军医大学西南医院全军消化病研究所,重庆400038 [2]第三军医大学全军免疫学研究所,重庆400038

出　　处：《重庆医学》2007年第9期820-822,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30570841)

摘　　要：目的预测肿瘤转移相关基因肝素酶的CTL表位,为探索基于肝素酶的抗原表位的免疫治疗奠定基础。方法利用基于超基序、量化基序结合人工神经网络方案开发的H-2Kb限制性CTL表位预测软件,对小鼠肝素酶蛋白的CTL表位进行预测,然后人工合成相关待测表位肽,再对这些合成肽与MHC-I结合亲和力进行检测。结果采用机算机网络系统,预测了5条小鼠肝素酶抗原表位,即mHpa234~241(LGEDFVEL),mHpa351~358(FAAGFMWL),mHpa519~526(FSYGFFVI),mHpa386~393(VDENFEPL),mHpa398~405(LSLLFKKL),MHC亲合力实验表明,与阴性对照肽对比,随着预测肽浓度增加,H-2Kb的表达逐渐增加,与MHC-Ⅰ类分子的亲和力亦逐渐增强,当预测肽浓度达到30μmol/L时,其荧光系数(FI)均大于1.5。结论通过计算机软件预测到的5条小鼠肝素酶CTL表位与MHC-Ⅰ类分子均有较高的结合亲和力,为该表位的下一步体内鉴定及基于小鼠肝素酶抗原表位的肿瘤免疫治疗奠定基础。Objiective To identify H-2K^b-restricted epitopes from a new cancer metastasis antigen heparanase. This work will contribute vastly to the application of specific immunotherapy on the basis of heparanase derived epitope. Methods Supermotif, quantitative motif and artificial neural network （ANN） were used in the prediction of H-2K^b restricted cytotoxic T lymphocyte （CTL） epitope from amino acid sequence of mouse heparanase. The peptides were synthesized with solid phase strategies,purified with reverse phase HPLC,identified and determined with mass spectrometry. RMA-S cell line was used to determine the peptide binding with H-2K^b molecule. Results Five candidate octopeptide [mHpa234-241 （LGEDFVEL）,mHpa351-358 （FAAGFMWL）,mH- pa519-526 （FSYGFFVI）, mHpa386-393 （VDENFEPL）, mHpa398-405 （LSLLFKKL） ] were predicted by supermotif combined with quantitative motif and ANN. These octopeptide peptides were above 95% in purity and the determined values of molecular weight conformed to their theoretical values. Further study showed that the expression of H-2K^b on the surface of RMA-S cell was increased consistly with the increase of predicted peptides. It is affinity with MHC-I molecule enhanced consistently. When predicted peptide concentration was more than 30μmol/L, it is fluorescent index（FI） all exceeded 1.5. Conclusion Epitope prediction with supermotif quantitative motif and ANN is consistent with peptide binding assay. In this study ,both peptide prediction and peptide binding assay showed that among five predicted epitopes, mHpa234-24t （ LGEDFVEL）, mHpa351-358 （ FAAGFMWL）, mHpa519 - 526 （ FSYGFF- VI）,mHpa386-393 （VDENFEPL） ,mHpa398-405 （LSLLFKKL） was determined as the most H-2K^b affinity.

关 键 词：鼠肝素酶 CTL表位 H-2K^b限制性 

分 类 号：R73-3[医药卫生—肿瘤]