【摘要】 目的:探讨采用最低限度免疫定义基因表达法制备丙型肝炎病毒(HCV)多表位DNA疫苗的可行性,阐明该方法在疫苗领域可能的应用价值。方法:采用人工合成和PCR方法制备长度为1 346bp的含有CMV启动子、HCV 1b亚型多表位和牛生长激素(BCG)多聚腺苷酸序列的最低限度免疫定义基因表达DNA疫苗,命名为M-HCV-epi;同时制备结构基因被非HCV同源的DNA序列替换的相同长度DNA片段作为对照,命名为V-pcDNA3.1。12只ICQ小鼠随机分为实验组(n=6)和对照组(n=6),分别采用M-HCV-epi DNA和V-pcDNA3.1DNA各20μg皮下注射。QRT-PCR法检测免疫后小鼠脾细胞中干扰素γ(IFN-γ)mRNA的表达水平;人工合成3个HCV 1b亚型表位多肽和1条对照多肽。用上述合成的多肽刺激免疫后小鼠脾细胞,ELISA法检测脾细胞刺激上清中IFN-γ水平。结果:与对照组比较,实验组小鼠脾细胞中IFN-γmRNA表达水平升高(1.50±0.18)倍(P<0.05);加入aa35-44多肽的实验组小鼠脾细胞培养上清中IFN-γ水平较对照组明显升高(P<0.05)。结论:最低限度免疫定义基因表达法制备HCV多表位DNA疫苗可诱导细胞免疫反应,该方法在DNA疫苗领域可能具有应用价值。更多还原

【Abstract】 Objective:To study the feasibility of hepatitis C virus(HCV)multi-epitope DNA vaccines prepared by minimalistic immunologically defined gene expression,and to clarify its potential application value in the field of DNA vaccines.Methods:A minimalistic immunologically defined gene expression DNA vaccine(1 346 bp)containing CMV promoter,HCV 1bsubtype multi-epitope,and bovine growth hormone(BCG)polyA sequence was prepared by artificial synthesis and PCR methods,then it was named M-HCV-epi.A control DNA fragment,which structure gene was replaced by a DNA fragment without homologous to HCV,was prepared and named VpcDNA3.1.A total of 12 ICQ mice were randomly divided into experimental group(n=6)and control group(n=6);the mice were subcutaneously injected with M-HCV-epi and V-pcDNA3.1 with the dosage of 20μg,respectively.The levels of interferon-γ(IFN-γ)mRNA in spleen cells of the immunized mice were detected by QRT-PCR method.A total of 3HCV 1bsubtype epitope polypeptides and a control polypeptide were synthetically prepared.The levels of IFN-γin culture supernatant of spleen cells were detected by ELISA method after the cells were stimulated with the polypeptides prepared above.Results:Compared with control group,the level of IFN-γmRNA in spleen cells of the mice in experiment group was increased to(1.50±0.18)times(P<0.05);compared with control group,the level of IFN-γin the culture supernatant of spleen cells of the mice in experiment group was increased(P<0.05).Conclusion:The HCV multi-epitope DNA vaccine with minimalistic immunologically defined gene expression can induce the cellular immune response,and may have application values in the field of DNA vaccines.更多还原