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目的:分析乙型肝炎病毒(HBV)重组多表位抗原基因在大肠杆菌中非融合表达的蛋白B-BPT的免疫原性,以期为HBV预防/治疗性疫苗的研制奠定实验基础。方法:构建重组质粒pBAD/BPT,并在大肠杆菌中诱导表达。以纯化的蛋白B-BPT免疫BALB/c小鼠,用乳酸脱氢酶(LDH)释放法检测该融合蛋白诱导的小鼠细胞毒T淋巴细胞(CTL)应答。用流式细胞术(FCM)检测小鼠CD4+、CD8+T淋巴细胞亚群的比例,用MTT比色法检测淋巴细胞的增殖效应。结果:B-BPT蛋白免疫BALB/c小鼠后,可诱导特异性CTL应答(P<0.05,与空白对照组比较);B-BPT蛋白免疫后,可显著刺激小鼠脾淋巴细胞增值(P<0.05,与空白对照组比较)。结论:该非融合表达的蛋白B-BPT可有效地诱导小鼠特异性CTL免疫应答,为进一步研制乙肝预防治疗性疫苗奠定了基础。
OBJECTIVE: To analyze the immunogenicity of non-fusion protein B-BPT of hepatitis B virus (HBV) recombinant multi-epitope antigen gene in E. coli and to lay the foundation for the development of HBV preventive / therapeutic vaccine. Methods: The recombinant plasmid pBAD / BPT was constructed and induced in E. coli. BALB / c mice were immunized with purified protein B-BPT and the cytotoxic T lymphocyte (CTL) responses induced by the fusion protein were detected by lactate dehydrogenase (LDH) release assay. The percentage of CD4 + and CD8 + T lymphocyte subsets in mice was detected by flow cytometry (FCM). The proliferation of lymphocytes was detected by MTT assay. Results: The specific CTL response was induced by B-BPT protein in BALB / c mice (P <0.05, compared with the blank control group); B-BPT protein immunization could significantly stimulate the proliferation of mouse spleen lymphocytes <0.05, compared with the blank control group). CONCLUSION: The non-fusion protein B-BPT can effectively induce specific CTL immune response in mice, which lays the foundation for further development of hepatitis B preventive vaccine.