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鸭源呼肠孤病毒DRV-GZ株S2基因的序列分析
及其表达
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刘红，郁宏伟，朱朝辉，吴志新，李苏芝，廖明*
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（华南农业大学 兽医学院，广东 广州510642）
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摘要：
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采用RT-PCR方法从鸭源呼肠孤病毒DRV-GZ株中扩增出了S2基因片段，将其克隆到表达载体pET-28a（+）中，测序验证后转化入表达宿主菌RosettaTM2（DE3）plysS，进行IPTG诱导表达。结果表明，重组菌可表达出相对分子质量约为50000的重组融合蛋白，在浓度为0.6mmol/L的IPTG诱导4 h的情况下表达效果最好。表达的蛋白以包涵体的形式存在于菌体中。表达产物经Ni柱纯化后可得到纯度较高的目的蛋白。经Western-blot分析，所纯化的蛋白能与抗呼肠孤病毒DRV-GZ株阳性血清进行特异性的免疫印迹反应，证实表达的蛋白具有较好的反应原性。 |
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关键词：
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鸭源呼肠孤病毒；S2基因；克隆；原核表达；纯化；免疫印迹 |
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中图分类号：S 852.659.4：Q 786 文献标识码：
A 文章编号：1673-4696(2008)03-0224-05 |
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Sequence analysis and expression of S2 gene of reovirus strain
DRV-GZ originated from duck |
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LIU Hong，YU Hong-wei，ZHU Chao-hui，WU Zhi-xin，LI Su-zhi，LIAO Ming |
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(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China) |
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Abstract: S2 gene of reovirus strain DRV-GZ originated from duck was amplified by RT-PCR and cloned into the pET-28a（+） expression vector.After sequencing,the recombinant plasmid was transformed into RosettaTM 2（DE3）plysS.The transformed bacteria were induced by IPTG and produced a recombinant protein of 50ku in mass. The results showed that 0.6mmol/L IPTG and 4 h of induction time were the optimal conditions for protein production and more pure proteins would be produced after purification with Ni+-column. The purified protein could react with the positive serum against strain DRV-GZ in a Western-blot test,indicating that the expressed protein had strong reactogenicity. |
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Key words: reovirus-originated duck;S2 gene;cloning;prokaryotic expression;purification；Western-blot |
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