| 摘要: |
| 通过利用分子生物学手段克隆IHHNV衣壳蛋白基因并构建原核表达载体，筛选抗原特性好的多肽免疫兔以制备高效的特异性IHHNV多克隆抗体，最终建立高效的检测对虾IHHNV的新型双抗夹心ELISA方法.实验结果表明该方法成功表达了对虾病毒IHHNV衣壳蛋白基因的抗原多肽，并制备出了高灵敏性的多克隆抗体.将抗原多肽及多克隆抗体用于ELISA的检测，抗体的效价在 1∶12 800 以上，最低可检测到1 pg的抗原蛋白，表明已成功建立高效的双抗夹心ELISA方法，对于对虾的海水养殖及IHHNV病毒性疾病的防控具有重要的参考价值. |
| 关键词: 海洋生物学 传染性皮下及造血组织坏死病毒 原核表达 衣壳蛋白 多克隆抗体 ELISA |
| DOI：10.3969/J.ISSN.2095-4972.2018.03.015 |
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| 基金项目:国家海洋局第三海洋研究所基本科研业务费资助项目（海三科2017005）；江苏省水产三新项目资助项目（D2015-11-5） |
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| Cloning and expression of IHHNV capsid protein and efficient detection of IHHNV through a new type of double antibody sandwich ELISA |
| YU Chen,LIU Guo-sheng,YANG Ming-han,YANG Li-rong,CHEN Ming-liang |
| (School of Marine Sciences, Ningbo University, Ningbo 315211, China; Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, SOA, Xiamen 361005, China) |
| Abstract: |
| In this study we used molecular biology methods to clone the gene of IHHNV capsid protein and construct prokaryotic expression vector. We screened the polypeptide with high antigen characteristics to immunize rabbits for preparing efficient and specific IHHNV polyclonal antibodies. Finally, we established a new highly efficient double antibody sandwich ELISA method to detect shrimp IHHNV. The results showed that the antigen polypeptide of the IHHNV capsid protein gene of shrimp virus was successfully expressed and highly sensitive polyclonal antibodies were prepared. When used the antigenic peptide and polyclonal antibodies for the detection of ELISA, the titer of ELISA was more than 1∶12 800, and the lowest antigen protein of 1pg could be detected. Our research showed that the highly efficient double antibody sandwich ELISA method has been successfully established, which can have important value for the shrimp culture and the prevention and control of IHHNV virus disease. |
| Key words: marine biology infectious hypodermal and hematopoietic necrosis virus prokaryotic expression capsid protein polyclonal antibody ELISA |
