| 含CSFV T细胞表位的猪细小病毒样颗粒的表达及小鼠试验免疫研究 |
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| 引用本文： | 范京惠,李一经.含CSFV T细胞表位的猪细小病毒样颗粒的表达及小鼠试验免疫研究[J].农业生物技术学报,2007,15(5). |
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| 作者姓名： | 范京惠 李一经 |
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| 作者单位： | 河北农业大学动物科技学院 东北农业大学 动物医学院 |
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| 摘 要： | 摘要：纯化的重组子pM-VP2-E290阳性质粒，与Bac-N-Blue DNA共转染昆虫细胞sf9，通过同源重组，形成具有感染活性的重组杆状病毒，并利用重组病毒的LacZ表型进行噬斑纯化。纯化后的高效价重组杆状病毒在昆虫细胞中进行表达，表达产物应用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）、免疫印迹（Western-blot）和免疫酶斑点技术（Dot-ELISA）进行分析，确定所表达的蛋白分子量大小约为67KD，且具有天然蛋白的抗原特异性。应用免疫电镜对表达产物进行观察，可见到细小病毒样颗粒。病毒样颗粒在无免疫佐剂参与的情况下，免疫6-8周龄的BALB/c小鼠，同时设PPV的灭活疫苗免疫组及PBS接种组作为参比对照，检测小鼠的细胞免疫和体液免疫学指标。结果表明，表达蛋白所形成的细小病毒样颗粒，不仅能诱导小鼠机体产生抗CSFV的特异性CTL反应，还能刺激小鼠产生高效价的抗PPV的特异性抗体。而且机体所产生的抗体效价显著高于灭活疫苗对照组。
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| 收稿时间： | 2007-2-14 |
| 修稿时间： | 2007-5-5 |
Expression and Immunogenicity of parvovirus-like particles |
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| Abstract: | Abstract: Purified PM-VP2-E290 and Bac-N-Blue DNA were cotransinfected insect cell sf9. The recombinant baculovirus were selected by their lacZ phenotypes and plaque-purified until no more wild type virus could be detected. Then, high-titer viral stocks of the recombinant baculovirus were prepared and used for expression. After SDS-PAGE analysis, an extra band of 67KDa was observed in the extracts of infected sf9 insect cells.The chimeric proteins had strong positive reactions with PPV specific antibody and E290-specific antibody. A large number of virus-like particles formed by expressed protein were observed under Immuno-electron microscopy (IEM).The BALB/c mice injected with the expressed protein, in the absence of adjuvant. showed that the hybrid recombinant parvovirus like-particles formed by the self-assembly of the VP2 capsid protein of PPV carring the CSFV epitope at its N terminal, not only induced a strong CD8+ class restricted CTL response, But stimulate a highly PPV specific antibody levels. Moreover, the antibody levels were significantly higher than that of the inactivated vaccine control. |
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