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

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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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    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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Ting GAO, Siyu WU, Caixia GAO, Sudong XIA, Jiyuan YIN, Yingying WANG, Yingying LI, Cunbin SHI, Qing WANG. Construction and identification of genotype Ⅱ grass carp reovirus virus-like particles[J]. Journal of Fisheries of China,2023,47(7):079413-1~079413-10

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History
  • Received:May 07,2021
  • Revised:August 31,2021
  • Adopted:September 16,2021
  • Online: July 11,2023
  • Published: July 01,2023
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