| 猪细小病毒NS1基因低温诱导表达及间接ELISA方法的建立 |
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| 引用本文： | 臧科伟,张春玲,邹勇,柴家前,崔言顺. 猪细小病毒NS1基因低温诱导表达及间接ELISA方法的建立[J]. 中国兽医学报, 2009, 29(10) |
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| 作者姓名： | 臧科伟 张春玲 邹勇 柴家前 崔言顺 |
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| 作者单位： | 1. 山东农业大学,动物医学院,山东,泰安,271018
2. 上海市农业科学院,畜牧兽医研究所,上海,201106 |
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| 基金项目： | 上海市农业科学院青年基金 |
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| 摘 要： | 利用PCR技术从猪细小病毒的DNA模板中扩增了NS1的基因片段,将PCR产物克隆至pET32c载体,将构建好的重组质粒pET32c-NS1,转入表达宿主茵BL21中,利用低温诱导表达的方法,避免了包涵体的形成.West-ern-blot结果显示,表达产物有良好的生物活性.纯化后的重组蛋白作为抗原,包被酶标板,建立了检测猪细小病毒特异性抗体的间接ELISA方法.抗原最佳包被质量浓度为2.5 mg/L、血清最佳稀释度为1∶200.表达的NS1蛋白可作为免疫诊断试剂用于检测PPV,且能很好的区别灭活疫苗免疫猪和自然感染猪.
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| 关 键 词： | 猪细小病毒 NS1基因 低温诱导 ELISA |
Prokaryotic expression by low-temperature and establishment of an indirect ELISA assay for the NS1 gene of porcine parvovirus |
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| ZANG Ke-wei,ZHANG Chun-ling,ZOU Yong,CHAI Jia-qian,CUI Yan-shun. Prokaryotic expression by low-temperature and establishment of an indirect ELISA assay for the NS1 gene of porcine parvovirus[J]. Chinese Journal of Veterinary Science, 2009, 29(10) |
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| Authors: | ZANG Ke-wei ZHANG Chun-ling ZOU Yong CHAI Jia-qian CUI Yan-shun |
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| Abstract: | The completed NS1 gene was amplified from PPV DNA by PCR method.Then the products were cloned into pET32c vector and the sequence was determined.The recombinant plasmid pET32c-NS1was transformed into BL21 competent cells and expressed in super-high level by using low-temperature induction to reduce formation of inclusion body.Western-blot analysis proved the renaturation protein has a good immunoreactivity against PPV antibody.The indirect ELISA method for the detection of PPV specific antibody in procine serum was established,after the optional working circumstances for the ELISA assay(antigen concentration:2.5 mg/L;optimal serum dilution:1∶200) with chessboard titration.The recombinant NS1 can be applied in differential diagnosis of PPV infections. |
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| Keywords: | porcine parvovirus gene NS1 low-temperature induction ELISA |
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