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目的构建以乙型肝炎病毒核心蛋白(Hepatitis B virus coreprotein,HBc)颗粒为载体的含流感病毒M2基质蛋白胞外功能区(M2 ectodomain,M2e)和核蛋白(Nucleoprotein,NP)保守表位的流感通用疫苗,评价其抗不同亚型流感病毒感染的保护作用。方法采用基因工程方法将流感病毒M2e的3拷贝重复片段与NP的CTL表位串联,插入HBc刺突顶端的免疫优势决定区,构建重组表达质粒pET-21a-HBc-3M2e-NP,转化大肠杆菌BL21(DE3),IPTG16℃低温诱导表达。表达的融合蛋白HBc-3M2e-NP经纯化后,电镜观察病毒样颗粒形成情况。纯化的融合蛋白分别经鼻腔和腹腔免疫小鼠,对照组注射等体积的PBS,间接ELISA法检测小鼠血清IgG抗体水平;流式细胞术检测小鼠脾组织中CD4+、CD8+T淋巴细胞水平;以流感病毒攻毒,检测融合蛋白的保护效果。结果重组表达质粒pET-21a-HBc-3M2e-NP经PCR及双酶切鉴定证明构建正确。表达的融合蛋白相对分子质量约为29000,以包涵体和可溶性两种形式表达,且可与鼠抗M2e单抗发生特异性反应。纯化的融合蛋白纯度大于90%,且能够自动装配成病毒样颗粒。用该病毒样颗粒免疫小鼠,可诱导小鼠产生针对不同流感病毒毒株的特异性抗体;2种途径免疫的小鼠脾组织中CD4+T淋巴细胞含量和CD4+/CD8+T淋巴细胞比例均较对照组明显升高;攻毒试验结果显示,具有交叉保护作用。结论构建的流感通用疫苗能够诱导机体产生高水平的体液免疫和细胞免疫应答及有效的交叉保护作用,为流感通用疫苗的深入研究奠定了基础。
OBJECTIVE: To construct influenza virus M2 M2 (M2 ect) and nucleoprotein (NP) conserved epitopes of influenza virus M2 (HBV) with Hepatitis B virus core protein (HBc) Universal vaccine to evaluate its protection against different subtypes of influenza virus infection. Methods Three replicative fragments of influenza virus M2e and CTL epitopes of NP were inserted into the immunodominance region at the top of HBc spike by genetic engineering to construct recombinant expression plasmid pET-21a-HBc-3M2e-NP and transformed into Escherichia coli BL21 (DE3), IPTG induced expression at low temperature of 16 ℃. The expressed fusion protein HBc-3M2e-NP was purified and observed for virus-like particle formation by electron microscopy. The purified fusion protein was immunized intranasally and intraperitoneally to mice respectively. The control group was injected with equal volume of PBS, and the level of IgG antibody in serum was detected by indirect ELISA. The levels of CD4 +, CD8 + T lymphocytes in spleen of mice were detected by flow cytometry ; Influenza virus challenge to test the protective effect of the fusion protein. Results The recombinant plasmid pET-21a-HBc-3M2e-NP was confirmed by PCR and restriction enzyme digestion. The expressed fusion protein has a relative molecular mass of about 29000, expressed in inclusion bodies and soluble forms, and can specifically react with mouse anti-M2e monoclonal antibody. The purified fusion protein is more than 90% pure and can be automatically assembled into virus-like particles. Immunization of mice with the virus-like particles induced mice to produce specific antibodies against different influenza virus strains. The levels of CD4 + T lymphocytes and CD4 + / CD8 + T lymphocytes in the spleen of 2 immunized mice Were significantly higher than the control group; challenge test results show that with cross-protection. Conclusion The universal influenza vaccine can induce the body to produce a high level of humoral and cellular immune response and effective cross protection, and lay the foundation for the further study of the universal flu vaccine.