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  

Induction of cellular immunity by HCV multi-epitope DNA vaccine prepared with minimalistic immunologically defined gene expression method in mice

作　　者：辛桂杰[1] 朱海超[2] 李昊[1] 温剑平[3] 

机构地区：[1]吉林大学第一医院肝胆胰内科,吉林长春130021 [2]吉林大学基础医学院法医学系,吉林长春130021 [3]吉林大学基础医学院遗传学系,吉林长春130021

出　　处：《吉林大学学报（医学版）》2016年第6期1071-1075,共5页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科研基金资助课题(20130413003GH)

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。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.

关 键 词：最低限度免疫定义基因表达 丙型肝炎病毒 多表位 DNA疫苗 干扰素Γ 

分 类 号：Q813[生物学—生物工程]