重组结核抗原痘苗病毒Ankara株的构建及其免疫原性研究

Construction of recombinant modified vaccinia virus Ankara with Ag85A and ESAT-6 gene and examination of their immunogenicity in mice

目的构建5种不同类型的表达结核杆菌特异抗原的重组痘苗病毒，并研究其特异免疫原性。方法运用同源重组技术将含结核分泌抗原Ag85A和ESAT-6的基因片段插入痘苗病毒表达质粒p18中。重组质粒导入痘苗病毒Ankara（MVA）后构建重组痘苗病毒，经筛选和Western blot鉴定，得到5个种类的带有结核抗原基因的重组病毒。用构建的5种重组病毒免疫小鼠，MTT法检测免疫后小鼠脾淋巴细胞对特异结核抗原的增殖反应；ELISA检测小鼠脾淋巴细胞培养上清液中IFN-γ的含量；结核菌素纯蛋白衍化物（PPD）皮内试验以检测重组病毒引发的针对结核抗原的特异细胞免疫应答。结果构建的5种重组病毒介导的细胞表达产物经Western blot鉴定确认相对分子质量与结核抗原一致。免疫小鼠两次后，5种重组病毒免疫组脾淋巴细胞体外与Ag85A—ESAT-6融合蛋白共培养后表现出明显的增殖活性（P〈0．01），培养上清液中IFN-γ的浓度均较同组细胞经生理盐水刺激明显增高（P〈0．05）；与空痘苗病毒或生理盐水免疫后小鼠相比，5种重组MVA免疫组脾淋巴细胞与Ag85A-ESAT-6融合蛋白共培养后同样表现出明显的增殖活性（P〈0．01），与Ag85A—ESAT-6融合蛋白共培养的细胞上清液中IFN-γ的浓度均升高（P〈0．01）。与空痘苗病毒或生理盐水免疫后小鼠相比，5种重组MVA免疫组小鼠对PPD都表现出显著的迟发型超敏反应应答（P〈0．05）。结论成功构建了5种不同类型的表达结核杆菌抗原的重组痘苗病毒疫苗，其免疫小鼠后可激发针对结核杆菌抗原的特异性细胞免疫。

Objective To construct five types of recombinant modified vaccinia virus Ankara （MVA） carrying genes encoding antigen 85A （Ag85A）, early secretory antigenic target （ESAT-6） or IL-2 and to investigate the immunogenicity of these recombinant MVA in mice. Methods The genes encoding Ag85A and ESAT-6 were amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA. The amplified DNA fragments were sub-cloned into vector p18. The recombinant plasmids were introduced respectively into MVA by homologous recombination. The five types of recombinant MVA clone were selected in the MPA selection medium and their expressions of TB antigens were confirmed by Western blot. The recombinant MVA were used to immunize BALB/c mice. Ag85A-ESAT-6 fusion protein induced proliferation of spleno-lymphocytes from immunized mice was detected by MTT test. The concentration of IFN-γ in supernatant of spleno-lymphocytes cultures was also analyzed by ELISA. The delayed type hypersensitivity （DTH） response to tuberculosis antigens was measured after injection of tuberculin purified protein derivative （PPD） in hind footpads of immunized mice. Results The five types of recombinant MVA were successfully constructed by confirming their expression of TB antigen with Western blot. After co-cultured with the fusion protein Ag85A-ESAT-6 in vitro, the spleno-lymphocytes from recombinant MVA immunized mice showed significant proliferation activity （ P 〈 0.01 ） and increased IFN-γ secretion （ P 〈 0.05 ） while the same cells showed no response when co-cultured with saline. The spleno-lymphocytes from recombinant MVA immunized mice also demenstrated significant proliferation activity （P 〈 0.01 ） and increased IFN-γ secretion （P 〈0.01 ） under Ag85A-ESAT-6 inducement, compared with the spleno-lymphocytes from mice immunized with wild type MVA or saline. The significant DTH response to PPD was detected in recombinant MVAimmunized mice （ P 〈 0.05 ）. Conclusion The five types of recombinant MVA carrying genes of TB antigens were constructed. The constructed recombinant MVA could induce specific cellular immunity against tuberculosis antigen in mice.