基因Ⅱ型草鱼呼肠孤病毒病毒样颗粒的构建与鉴定
作者:
作者单位:

1.天津农学院水产学院,天津 300384;2.中国水产科学研究院珠江水产研究所,农业农村部渔药创制重点实验室,广东省水产动物免疫技术重点实验室

作者简介:

通讯作者:

王庆,从事水产病害防治研究,E-mail:wangqing@prfri.ac.cn

WANG Qing. E-mail: wangqing@prfri.ac.cn

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基金项目:

国家重点研发计划 (2019YFD0900103);中国水产科学研究院基本科研业务费 (2021SJ-XK1);国家大宗淡水鱼产业技术体系 (CARS-45);广东省现代农业产业技术体系创新团队建设专项 (2021KJ150)


Construction and identification of genotype Ⅱ grass carp reovirus virus-like particles
Author:
Affiliation:

1.Department of Aquaticture, Tianjin Agricultural University, Tianjin 300384, China;2.Key Lab of Fishery Drug Creation of Ministry of Agriculture and Rural Affairs, Key Lab of Aquatic Animal Immune Technology of Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China

Fund Project:

National Key Research and Development Program of China (2019YFD0900103); Central Public-interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences (2021SJ-XK1); China Agriculture Research System (CARS-45); Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2021KJ150)

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    摘要:

    为研究Ⅱ型草鱼呼肠孤病毒 (GCRV)病毒样颗粒 (VLPs)疫苗,实验利用杆状病毒-昆虫细胞表达系统 (BEVS)将具有免疫原性的GCRV-VP3/VP4/VP35蛋白进行GCRV-VLPs的组装。实验将编码VP35蛋白的GCRV-s11基因克隆入杆状病毒载体pFastBacHTATM,然后将鉴定正确的重组质粒转化至DH10Bac感受态细胞,筛选得到重组穿梭质粒Bacmid-VP35。将穿梭质粒Bacmid-VP35以及实验室前期构建的重组穿梭质粒Bacmid-VP3、Bacmid-VP4分别转染Sf9昆虫细胞获得重组杆状病毒pFHB-VP35、pFHB-VP3以及pFHB-VP4。利用Bac-PAK快速滴定试剂盒测定重组杆状病毒滴度,并通过间接免疫荧光 (IFA)和蛋白质印迹法 (Western blot)鉴定重组蛋白的表达情况。结果显示,实验获得了较高滴度的重组杆状病毒,并且重组蛋白在杆状病毒感染的Sf9昆虫细胞中正确表达。将成功表达蛋白的重组杆状病毒pFHB-VP35、pFHB-VP3以及pFHB-VP4共感染Sf9细胞组装GCRV-VLPs,通过透射电镜 (EM)观察VLPs的组装情况。结果显示,GCRV-Ⅱ的3个蛋白在Sf9昆虫细胞中可以完成自我组装,形成与天然病毒结构形似的VLPs,直径为65~72 nm。本实验结果为进一步研制安全、高效的GCRV-VLPs疫苗奠定了基础。

    Abstract:

    In order to construct genotype II grass carp reovirus (GCRV) virus-like particulars (VLPs) based vaccines, the immunogenic GCRV-VP3/VP4/VP35 protein was assembled into GCRV-VLPs using baculovirus expression system (BEVS). The structural protein VP35 encoded by the segment S11 of GCRV-II was inserted into baculovirus vector pFastBacHTATM using BEVS, and the identified recombinant vector was transformed into DH10Bac competent cells to screen recombinant plasmid Bacmid-VP35. Recombinant baculovirus pFHB-VP35 was obtained by transfecting recombinant plasmid Bacmid-VP35, recombinant baculovirus pFHB-VP3 and pFHB-VP4 was obtained by transfecting recombinant plasmid Bacmid-VP3 and Bacmid-VP4 which have been successfully constructed in our laboratory. Then the titers of recombinant baculovirus were determined by Bac-PAK rapid titer kit and the expression of proteins was identified by IFA and Western blot. The results showed that high titers of pFHB-VP35, pFHB-VP3, and pFHB-VP4 were obtained, and the corresponding proteins were successfully expressed in infected Sf9 cells. GCRV-VLPs were assembled co-infection of Sf9 cells with pFBH-VP35, pFBH-VP3, and pFBH-VP4, and the GCRV-VLPs were identified by electron microscopy (EM). The results showed that the three proteins could self-assemble in Sf9 cells and formed VLPs with diameter of 65-72 nm similar with natural viruses. This study lays a foundation for development of novel vaccines for preventing the disease caused by GCRV genotype II.

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郜婷,吴斯宇,高彩霞,夏苏东,尹纪元,王英英,李莹莹,石存斌,王庆.基因Ⅱ型草鱼呼肠孤病毒病毒样颗粒的构建与鉴定[J].水产学报,2023,47(7):079413-1~079413-10

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  • 收稿日期:2021-05-07
  • 最后修改日期:2021-08-31
  • 录用日期:2021-09-16
  • 在线发布日期: 2023-07-11
  • 出版日期: 2023-07-01