利用IFNγ - ELISPOT评价肝癌复合表位抗原细胞免疫反应.docVIP

利用IFNγ - ELISPOT评价肝癌复合表位抗原细胞免疫反应 　【摘要】 目的 构建肝癌多表位基因和含Tat PTD的多表位基因的原核表达载体，表达纯化融合蛋白，比较其细胞免疫反应。方法 人工合成多表位和Tat基因；分别克隆入原核表达载体pBVIL1，构建含Tat PTD及不含Tat PTD的肝癌多表位基因原核表达载体pBVIL1/Tat+T、pBVIL1/T；诱导表达、纯化目的蛋白；IFNγ- ELISPOT法检测免疫小鼠的细胞免疫应答。结果 正确构建复合表位基因的原核表达质粒，目的蛋白质在大肠杆菌中获到高效表达，并得到有效纯化；ELISPOT检测显示Tat+T蛋白免疫组能更有效地激发特异性T细胞免疫反应。结论 所构建的含Tat PTD的新型复合表位抗原能激活特异性CTL反应，为基于CTL表位的肝癌免疫治疗提供了实验依据。 【关键词】 肝细胞癌 T细胞表位 ELISPOT Tat PTD 【Abstract】 Objective To construct the prokaryotic recombinant plasmid to express HCC multiple epitopes gene, then purify the fused protein for ELISPOT analysis.Methods The gene of multi-epitope were gained and it was cloned into prokaryotic expression vector and expressed in E.coli. An recombinant eukaryotic expression vector was constructed and its eukaryotic expression was determined in transfected cells. Balb/c mice were immunized with different forms of antigen. The cell immune response was tested by ELISPOT.Results The recombinant prokaryotic plasmid was correctly constructed. The gene was expressed in E. coli. highly and the expressed protein was purified efficiently. The antigen specific T cell response IFN-γ release was much stronger in protein Tat+T group than in the other groups.Conclusion This preliminary result showed that immunization with those improved recombinant polyepitope antigen has high antigenicity, especially in induction of high CTL response. This research may be useful to further development of immunotherapy for HCC. 【Key words】 hepatocellular carcinoma;T-cell epitopes;ELISPOT;TatPTD 肝细胞癌（HCC）是世界上最常见的恶性肿瘤之一，我国肝癌患者约占全世界的50%，在我国加强肝癌的防治研究意义重大。随着分子免疫学、肿瘤免疫治疗学的迅猛发展，使免疫治疗可能成为治疗肝癌的有效手段［1～3］。主动特异性免疫治疗即肿瘤疫苗因其特异性高、针对性强又成为免疫治疗的方向与主流。寻找肿瘤特异性/相关性抗原，增强其免疫原性，提高肿瘤免疫治疗的效果已经成为肿瘤疫苗研究的核心问题。因此，本研究拟采用肝癌中高表达的AFP、MAGE-1分子作为模式肿瘤抗原，从抗原递呈方式角度提高肝癌复合表位的免疫原性，为基于CTL表位肽的肝癌免疫治疗打下基础。 1 材料与方法 1.1 材料 DNA限制性内切酶（EcoRⅠ、BamHⅠ、XhoⅠ和XbaⅠ）；PCR产物纯化试剂盒和质粒提取试剂盒购自博大生物公司；原核表达载体pET22b为Novagen公司产品，原核表达载体pBVIL1由本室构建并保存［4］；Ni-NTA superflow 购自 QIAGEN公司；ELISPOT检测采用法国DIACLONE公司的Murine IFNγ ELIS