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2025年4月6日 星期日
  2016, Vol. 24 Issue (9): 1346-1353    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
猪圆环病毒2 d亚型衣壳蛋白在昆虫细胞中的表达
黄娟,袁飞,韩先杰,李桂梅,单虎
青岛农业大学
Expression of Porcine Circovirus Subtype 2 d Capsid Protein in Baculovirus/insect Cell Expression System
2, 2, 2, 2
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摘要 为了在昆虫细胞中表达猪圆环毒病2型(Porcine circovirus type 2, PCV2)衣壳蛋白,本研究设计合成引物从临床病料中扩增获得猪圆环病毒(Porcine circovirus, PCV)山东株衣壳蛋白基因缺失核定位区的序列,并进行序列分析。将该片段克隆到杆状病毒供体载体pFastBac-1上,然后转化大肠杆菌(Escherichia coli)DH10-Bac感受态细胞,获得重组穿梭杆粒,将该杆粒转染到sf9昆虫细胞中,观察重组杆状病毒致细胞病变效应,并采用PCR、间接免疫荧光实验、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)和Western blot鉴定衣壳蛋白的表达情况。结果表明,扩增获得的猪圆环病毒山东株属于PCV2d亚型,与我国目前使用的商品化疫苗株(PCV2a及PCV2b亚型)在160~180 aa抗原位点有差异。成功构建了重组供体质粒pFastBac1-PCV2d-Cap和重组穿梭杆粒Bacmid-PCV2d-Cap,获得的重组杆状病毒在昆虫细胞中成功表达了约24 kD的PCV2d-Cap,该蛋白可以与猪圆环病毒阳性血清及His标签抗体发生反应,证明具有良好的反应原性。本研究是我国首次采用杆状病毒系统表达PCV2d亚型衣壳蛋白,为制备PCV2d亚单位疫苗以及酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)试剂盒、防范PCV2d亚型流行提供了理论依据。
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黄娟
袁飞
韩先杰
李桂梅
单虎
关键词 猪圆环病毒2d亚型衣壳蛋白杆状病毒表达系统基因分型    
Abstract:In order to express capsid protein of porcine circovirus type 2 (PCV2) in insect cells, nuclear-localization-signal (NLS) free capsid protein gene of PCV2 Shandong strain was amplified from clinical samples, then, the gene was inserted into baculovirus transfer vector pFastBac-1, the resulted plasmid was transferred to competent Escherichia coli DH10-Bac cells, the recombinant shuttle Bacmid was transfected into sf9 insect cells and cytopathic effect was observed. Capsid protein expression was identified by PCR, indirect immunofluorescence test, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The results showed that a 614 bp-length fragment was amplified from suspected samples, the amplified capsid protein gene of PCV2 Shandong strain shared highest homology with PCV2d strains at nucleotide level (99.8%) and amino acid level(100%), and belong to PCV2d subtypes revealed by evolutionary tree. Comparing with vaccine strains (PCV2a or PCV2d) widely used in China, this PCV2d strain showed a different antigenic domain at 160~180 aa. pFastBac1-PCV2d- Cap and Bacmid-PCV2d-Cap were successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. The recombinated baculovirus induced cytopathogenic effect in sf9 insect cells, and successfully expressed a 24 kD protein, which could react with PCV2 antiserum and His-tag monoclonal antibody. The good immunogenicity of expressed capsid protein provides possible application in the development of porcine circovirus virus type 2d subunit vaccine and ELISA kit. This is the first report about PCV2d capsid protein expressed in baculovirus/insect cell expression system.
Key wordsPorcine circovirus subtype 2 d    Capsid protein    Baculovirus expression    Genetic type
收稿日期: 2016-03-25      出版日期: 2016-07-22
基金资助:国家科技基础性工作专项项目;山东省现代农业产业技术体系生猪产业创新团队疫病控制岗位专家
通讯作者: 单虎     E-mail: hush@qau.edu.cn;shanhu67@163.com
引用本文:   
黄娟 袁飞 韩先杰 李桂梅 单虎. 猪圆环病毒2 d亚型衣壳蛋白在昆虫细胞中的表达[J]. , 2016, 24(9): 1346-1353.
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http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I9/1346
 
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