首页|G-四链体碱基突变可增强丙型肝炎病毒DNA疫苗的免疫原性

G-四链体碱基突变可增强丙型肝炎病毒DNA疫苗的免疫原性

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目的 探讨丙型肝炎病毒(Hepatitis C Virus,HCV)核心抗原core基因上的G4-四链体(core G4)RNA结构对机体特异性免疫应答的影响.方法 圆二色谱(circular dichroism,CD)检测寡核苷酸链RNA G4(命名为G4R)和突变体G4M(命名为G4RM,在不改变氨基酸密码子的情况下)的G-四链体空间结构;在不改变密码子的情况下采用PCR定点突变的方法把HCV野生型G4-core(DNA)突变为G4M-core,然后将G4-core和G4M-core基因构建至真核表达质粒pcDNA3.1上(分别称为pG4和pG4M),免疫印迹(Western blot)分析core基因上G4结构突变后Core蛋白的表达水平.将pG4及pG4M质粒分别用基因导入仪免疫小鼠,检测免疫后小鼠的细胞免疫以及体液免疫水平.结果 CD结果显示寡核苷酸链RNA G4M相比野生型RNA G4结构发生改变,熔解曲线表明G4RM熔解温度低于野生型RNA G4R.Western blot显示在细胞水平及动物水平,pG4M比pG4的Core蛋白表达水平更高(P<0.05).动物免疫实验结果表明pG4M免疫后小鼠总IgG及IFN-γ的释放水平增高(P<0.05).其中,pG4M免疫组小鼠的IgG水平是pG4组的1.61倍.酶联免疫斑点(Enzyme-linked immunospot,ELISpot)实验中,pG4M免疫后小鼠脾脏细胞IFN-γ的释放水平是pG4的1.39倍.流式实验显示pG4M免疫后小鼠脾脏CD4+T细胞中IFN-γ产生水平是pG4的1.79倍.结论 本研究首次报道HCV核心抗原core上G-四链体可能抑制其蛋白翻译和免疫应答,core上G-四链体碱基突变可增强翻译水平,并增强其Th1型的细胞免疫应答.核心抗原G-四链体碱基突变可增强HCV疫苗的免疫原性,为研制增强免疫原性的HCV疫苗提供了一种新的策略.
The G4-quadruplex mutation of the core gene enhances the immunogenicity of hepatitis C virus DNA vaccine
Objective To investigate the effect of G-quadruplex (G4) RNA structure of core of hepatitis C virus (HCV) on the specific immune response. Methods Circular dichroism (CD) was usedto detect the G4 spatial structure of the G4 oligonucleotide chain RNA (named as G4R) and its mutant of G4 (named as G4RM) by G base site-specific mutation.The HCV wild-type core gene G4(DNA) sequence was mutated as G4M-core by PCR site-directed mutagenesis without changing the amino acid codon.Then wild type and mutated core genes were constructed into the eukaryotic expression plasmid pcDNA3.1-Myc, and produced as pcDNA3.1-core-G4-WT (named as pG4) and pcDNA3.1-core-G4-M (named as pG4M), the expression of core protein was examined by Western blot. The mice were immunized with the pG4 and pG4M plasmids DNA respectively, and their humoral and cellular responses were examined. Results CD results showed that the structure of G4RM was changed compared to Wild type G4R, and the melting curve analysis showed the melting temperature of GR4M was lower than that of G4R, which indicates that G4RM structure is unstable. Western blot analysis showed that pG4M had much higher protein expression level compared to pG4(P<0.05). Analysis of animal immunization showed that pG4M induced increased levels of total IgG and IFN-γ compared to pG4(P<0.05). The IgG level of the pG4M group was 1.61 times higher than that of the pG4 group. By enzyme-linked immunospot(ELISpot)assay, we found that the release IFN-γ level of pG4M was 1.39 times higher than those of pG4. Flow cytometry showed that the intracellular IFN-γ production in the splenic CD4+ T cells was 1.79 times than those of pG4. Conclusion The G-quadruplex structure of HCV core can inhibit its protein translation. The mutation of G-quadruplex of core led to increased Th1-type immune responses. This is the first report demonstrate that HCV core G-quadruplex mutation can enhance its immunogenicity and could be used as a new strategy ofexploring HCV vaccine with enhanced immunogenicity.

hepatitis C virus coreG-quadruplexpoint mutationimmune response

王昱斌、卞文秀、刘敏、章晓联

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武汉大学病毒学国家重点实验室/湖北省过敏及免疫相关疾病重点实验室/武汉大学 医学研究院/武汉大学基础医学院 免疫学系,湖北 武汉 430071

核心抗原Core G-四链体 点突变 免疫应答 细胞免疫

国家自然科学基金国家自然科学基金国家自然科学基金国家杰出青年基金湖北省医学领军人才计划湖北省技术创新专项重大项目湖北省技术创新专项重大项目武汉科技项目

21572173313701978150137781025008523-2760032016ACA1502016CFA062201150530141

2017

10.3969/j.issn.1005-1678.2017.01.002

中国生化药物杂志
南京生物化学制药研究所,全国生化制药情报中心站,中国生化制药工业协会,中国药品生物制品检定所

中国生化药物杂志

ISSN：1005-1678
年,卷(期)：2017.37(1)
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