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
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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.
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.
引文
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[2]Perez Ruiz,Garibay A.Endocytosis in gene therapy with non-viral vectors[J].Wien Med Wochenschr,2016,2(3):556-562.
[3]Pergament E.The promise of gene therapy[J].Curr Opin Obstet Gynecol,2016,28(2):132-135.
[4]Machelska H,Schroff M,Oswald D,et al.Peripheral nonviral MIDGE vector-driven delivery ofβ-endorphin in inflammatory pain[J].Mol Pain,2009,5(6):72-85.
[5]López-Fuertes L,Pérez-Jiménez E,Vila-Coro AJ,et al.DNA vaccination with linear minimalistic(MIDGE)vectors confers protection against Leishmania major infection in mice[J].Vaccine,2002,21(4):247-257.
[6]Zheng C,Juhls C,Oswald D,et al.Effect of different nuclear localization sequences on theimmune responses induced by a MIDGE vector encoding bovine herpesvirus-1glycoprotein D[J].Vaccine,2006,24(21):4625-4629.
[7]Chen ZY,He CY,Ehrhardt A,et al.Minicircle DNA vectors devoid of bacterial DNA result in persistent and highlevel transgene expression in vivo[J].Mol Ther,2004,8(3):495-500.
[8]Wei L,Han T,Yang D,et al.Simeprevir plus peginterferon/ribavirin for HCV genotype 1-infected treatment-nave patients in China and South Korea[J].J Gastroenterol Hepatol,2016,31(5):912-920.
[9]Christiane L,Schnabel P,Steinig M,et al.Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent[J].BMC Vet Res,2015,11(10):140-144.
[10]Agarwal A,Ingham SA,Harkins KA.The role of pharmacogenetics and advances in gene therapy in the treatment of diabetic retinopathy[J].Pharmacogenomics,2016,17(3):309-320.
[11]Pahle J,Walther W.Vectors and strategies for nonviral cancer gene therapy[J].Expert Opin Biol Ther,2016,16(4):443-461.
[12]Campbell JP,McFarland TJ,Stout JT.Ocular gene therapy[J].Dev Ophthalmol,2016,55(3):317-321.
[13]韩笑,麻树人,王大全,等.早期骨癌外周血细胞免疫功能状况变化研究[J].中国实用内科杂志,2015,35(9):759-761.
[14]李修明.基于DC-CIK细胞免疫治疗联合化疗治疗晚期消化道肿瘤的临床疗效分析[J].中国实用内科杂志,2015,35(增1):119-121.