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口蹄疫病毒3B基因的高效表达及其表达产物
的反应活性
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覃健萍1，3,徐维加2,陈峰1，3,马静云1,谢青梅1,刘盛梅1,曹永长1,毕英佐1
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（1.华南农业大学 动物科学学院，广东 广州510640；2.云南出入境检验检疫局，云南 昆明650228；
3.广东温氏食品集团有限公司，广东 新兴527439）
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摘要：
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通过PCR技术获得口蹄疫病毒非结构蛋白3B基因，将其克隆至质粒pET-32a(+)和高效可溶性表达质粒pEM中，经PCR筛选，获得阳性重组子pET-3B和pEM-3B，将它们转化大肠杆菌BL21中进行诱导表达，SDS-PAGE及Western-blot检测结果证明，表达的3B融合蛋白能与FMDV感染动物血清发生特异性反应。蛋白可溶性分析表明，EM-3B融合蛋白绝大部分以可溶形式表达。以此可溶性融合蛋白EM-3B为抗原建立了可区分FMD免疫动物和感染动物的EM-3B-DIVA-ELISA方法，经检测，其特异性为950%，敏感性为97.5%，与2个国产试剂盒的符合率分别为96.25%和98.75%。 |
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关键词：
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口蹄疫病毒；3B基因；高效可溶性表达；鉴别诊断ELISA |
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中图分类号：S 852.659.6：Q 786
文献标识码：
A 文章编号：1673-4696(2007)10-0867-06 |
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High expression of foot-and-mouth disease virus 3B gene and
reactivity of the expressed protein |
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QIN Jian-ping1，3,XU Wei-jia2,CHEN
Feng1，3,MA Jing-yun1,XIE Qing-mei1,
LIU Sheng-mei1,CAO Yong-chang1,BI Ying-zuo1 |
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（1.College of Animal Science,South China Agricultural University,Guangzhou 510640,China；
2.Yunnan Entry-Exit Inspection and Quarantine Bureau,Kunming 650228,China；
3.Guangdong Wen’s Foodstuffs Group Limited Company,Xinxing 527439,China） |
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Abstract: 3B gene encoding non-structural protein 3B of food-and-mouth disease virus(FMDV) was amplified by PCR and inserted into expression plasmids pET-32a(+) and pEM.The positive recombinants pET-3B and pEM-3B were screened by PCR and then transformed into Escherichia coli BL21 induced by IPTG.Analyses by SDS-PAGE and Western-blotting showed that the soluble EM-3B fusion protein expressed could react specifically with sera from animals infected with FMDV.Then,with the fusion protein EM-3B as a diagnostic antigen,an ELISA to differentiate FMDV-infected animals from vaccinated animals(EM-3B-DIVA-ELISA) was established.Results of specificity and sensitivity evaluations confirmed that the specificity and sensitivity of EM-3B-DIVA-ELISA were 95.0% and 97.5%,respectively.The coincidence rates of the EM-3B-DIVA-ELISA with the two commercial ELISA kits were 96.25% and 98.75% respectively. |
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Key words: foot-and-mouth disease virus;3B gene;solution expression with high efficiency;DIVA-ELISA |
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