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目的 :构建表达柯萨奇病毒B3(CVB3)主要包膜蛋白VP1的基因疫苗 ,并研究该疫苗诱导CVB3特异性免疫应答及免疫保护的作用。方法 :抽提CVB3RNA ,以RT PCR扩增VP1基因 ,克隆于真核表达载体 pcDNA3中 ,构建质粒pcDNA3 VP1。将该质粒转染Hela细胞 ,观察其表达情况 ;以 5 0 μgpcDNA3 VP1质粒DNA肌注免疫BALB/c小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答。间隔 4wk以 5×LD50 的CVB3攻击小鼠 ,观察攻击后小鼠的存活情况。结果 :构建了重组质粒 pcDNA3 VP1,并在体外获得有效表达。以该质粒肌肉免疫BALB/c小鼠 ,可诱生高水平的IgM和IgG ,VP1多肽特异性淋巴细胞增殖反应及CTL活性均显著高于 pcDNA3免疫的对照组。病毒攻击试验表明 ,pcDNA3 VP1免疫组33.3%小鼠可长期存活 ,其心肌组织未见明显的病理学改变 ;而对照小鼠平均仅存活 6 .7d ,心肌显示大量的局灶性坏死和炎性细胞浸润。结论 :pcDNA3 VP1免疫可诱生CVB3特异性体液及细胞免疫应答 ,保护免疫小鼠抵抗CVB3的致死性攻击
OBJECTIVE: To construct a gene vaccine expressing the major envelope protein VP1 of Coxsackievirus B3 (CVB3) and to investigate the effect of the vaccine on CVB3-specific immune response and immune protection. Methods: The CVB3 RNA was extracted and the VP1 gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3 to construct the plasmid pcDNA3 VP1. The plasmid was transfected into Hela cells to observe its expression. BALB / c mice were immunized three times with 50 μg pcDNA3 VP1 plasmid DNA to detect CVB3-specific humoral and cellular immune responses. Mice were challenged with 5 × LD50 of CVB3 at 4wk intervals to observe the survival of challenged mice. Results: Recombinant plasmid pcDNA3 VP1 was constructed and expressed in vitro. Immunization of BALB / c mice with this plasmid resulted in high levels of IgM and IgG, and the specific lymphocyte proliferation response and CTL activity of VP1 polypeptide were significantly higher than that of pcDNA3-immunized control group. Virus challenge test showed that 33.3% of mice immunized with pcDNA3 VP1 could survive for a long time, and no obvious pathological changes were found in the myocardium of mice. On the average, the control mice survived only 6.7 days, and the myocardium showed a large number of focal necrosis and inflammation Cell infiltration. CONCLUSION: Vaccination with pcDNA3 VP1 induces CVB3-specific humoral and cellular immune responses and protects immunized mice against lethal challenge with CVB3