最低限度免疫定义基因表达的HCV多表位DNA疫苗对小鼠细胞免疫的诱导作用

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1346 bp 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-pcDNA3.1 DNA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。%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 1b subtype 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 V-pcDNA3.1. 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 20 μg, 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 3 HCV 1b subtype 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.