鼠Ⅲ型肝炎病毒H-2K^K限制性CTL表位的初步鉴定

Primary identification of H-2K^K-restricted CTL epitopes from mouse hepatitis virus

目的预测和初步鉴定鼠Ⅲ型肝炎病毒(MHV-3)的CTL表位,为基于慢性肝炎病毒抗原表位的免疫治疗奠定理论基础。方法利用超基序、量化基序及人工神经网络方案相结合预测MHV-3 S蛋白的H-2K^K限制性CTL表位;用分子模拟对候选表位肽做进一步筛选;人工合成相关待测表位肽,利用T2-K^K细胞株测定各肽与H-2K^K分子的结合力。结果结合超基序、量化基序、人工神经网络预测结果及分子模拟结果,预测出了4条候选表位肽。MHC亲和力实验表明,在候选的4条表位肽中,IEPYNGVI(141-148)、YELSGYTV(306-313)与H-2K^K呈中等亲和力结合,荧光系数分别为1.25及1.04。结论表位预测的结果与亲和力分析结果一致性较好,两者联合应用初步认为IEPYNGVI(141-148)及YELSGYTV(306-313)为MHV-3 S蛋白H-2K^K限制性CTL表位的可能性最大,为下一步体内鉴定及基于小鼠肝炎病毒抗原表位的免疫治疗奠定基础。

Objective We identified H-2K^K-restricted CTL epitopes from mouse hepatitis virus strain 3, and the present study would benefit to the application of specific immunotherapy based on hepatitis virus derived epitope. Supermotif, quantitative motif combined with artificial neural network （ANN） and molecular modeling were used in the prediction of H-2K^K restricted cytotoxie T lymphocyte （CTL） epitope front amino acid sequence of mouse hepatitis virus strain 3. The predicted peptides were synthesized with solid phase strategies, purified with reverse phase HPLC, and finally identified with mass spectrometry; T2-K^K cell line was used to de- termine the peptide binding with H-2K^K molecule. At last, four candidate peptides were predicted by supermotif combined with quan- titative motif and ANN. Among the four predicted peptides, IEPYNGVI （141-148） and YELSGYTV （306-313） could bind to H-2K^K with moderately affinity, and the value of fluorescence index （FI） was 1.25 and 1.04, respectively. Our results suggested that epitope prediction with supermotif combined with quantitative motif and ANN was consistent with peptide binding assay. In this study, both epitope prediction and binding assay showed that IEPYNGVI （141-148） and YELSGYTV （306-313） may be the H-2K^K-restrieted epitopes from MHV-3.