文章摘要
牡蛎疱疹病毒囊膜蛋白(orf111)基因的克隆及表达*
Gene cloning and expression of Ostreid herpesvirus 1 envelope protein (orf111)
投稿时间:2019-07-09  修订日期:2019-07-31
DOI:
中文关键词: 牡蛎疱疹病毒  跨膜蛋白  原核表达;魁蚶
英文关键词: Ostreid herpesvirus 1  envelope protein  prokaryotic expression  Scapharca broughtonii
基金项目:现代农业产业技术体系建设专项资金,农业部重点实验室开放基金,中国水产科学研究院黄海水产研究所基本科研业务费
作者单位E-mail
张淑敏 大连海洋大学水产与生命学院 辽宁 大连 zhangshumin1223@foxmail.com 
白昌明 中国水产科学研究院黄海水产研究所  
辛鲁生 中国水产科学研究院黄海水产研究所  
李亚楠 中国水产科学研究院黄海水产研究所  
李晨 中国水产科学研究院黄海水产研究所  
王崇明① 中国水产科学研究院黄海水产研究所 wangcm@ysfri.ac.cn 
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中文摘要:
      牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)是一种具有囊膜的病毒,囊膜蛋白在病毒侵染宿主细胞的过程中起着决定性的作用。由于缺乏对该病毒敏感的细胞系,OsHV-1结构蛋白的定位和功能研究进展缓慢。本研究对编码OsHV-1囊膜蛋白的orf111基因进行了生物信息学分析、克隆和表达。首先通过特异性PCR扩增克隆得到orf111基因编码框全长序列,并对其编码囊膜蛋白的理化性质、高级结构、跨膜区及抗原决定簇等进行生物信息学分析。结果显示,orf111编码一种稳定的疏水性蛋白,具有5个跨膜结构域,9个抗原决定簇,同时氨基酸序列中还包含一个高度保守的精氨酰-甘氨酰-天冬氨酸(Arg-Gly-Asp, RGD结构域。随后成功构建了pET28a-orf111重组质粒,并将其转化到大肠杆菌DH5α感受态细胞中;最后通过使用异丙基-β-D-硫代半乳糖苷(IPTG)成功诱导蛋白表达,表达产物分子量约为32 kDa。本研究应用原核表达技术成功得到含RGD结构域的OsHV-1囊膜蛋白,为进一步制备ORF111蛋白单克隆抗体及开展牡蛎疱疹病毒侵染机制的研究奠定了重要基础,为将来OsHV-1的防控工作提供新的思路。
英文摘要:
      Ostreid herpesvirus 1 (OsHV-1) is a kind of virus with capsule membrane. The virus envelope proteins plays a decisive role in the process of host cells. Nowadays, the localization and function of structural proteins of the OsHV-1 are progressing slowly because lack of the cell lines sensitive to the virus. In this study, we studied the envelope protein (ORF111) in OsHV-1, like bioinformatics analysis, gene cloning and protein expression. Firstly, we designed the primers of orf111 gene based on the complete genome sequence of OsHV-1, and successfully cloned the OsHV-1-orf111 gene. We used some bioinformatics methods to predict and analyzed the gene sequence and the characteristics of the protein encoded by the OsHV-1-orf111 gene, the characteristics of the ORF111 protein including the physicochemical properties, advanced structure, trans-membrane region, antigen determinant cluster and so on. According to the bioinformatics methods analysis results, the ORF111 protein is a stable hydrophobic protein, the amino acid sequence contains a highly conservative pure ammonia Arginyl-Glycyl-Aspartate (Arg-Gly-Asp, RGD) sequence, and there are five transmembrane domain structures, and nine antigenic determinants. Then the OsHV-1-orf111 gene was linked with pET28a(+) plasmid, and the recombinant plasmid pET28a-orf111 was successfully constructed. After the recombinant plasmid was transformed into DH5α, the protein expression was induced by isopropyl-beta-d-thiogalactosine (IPTG). The SDS-PAGE test showed that a large number of ORF111 protein was successfully expressed, and the molecular weight of ORF111 protein was about 32 kDa. In this study, OsHV-1 envelope protein containing RGD domain was successfully obtained by prokaryotic expression technique, which laid a certain foundation for the preparation of monoclonal antibody and functional study of ORF111, and provided a basis for further study on the infection mechanism of OsHV-1. And provided a new idea for the prevention and control of OsHV-1 in the future.
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