title: Protective immunity in gibel carp, Carassius gibelio of the truncated proteins of cyprinid herpesvirus 2 expressed in Pichia pastoris.
abstract: Cyprinid herpesvirus 2 (CyHV-2) infection is a newly emerged infectious disease of farmed gibel carp (Carassius gibelio) in China and causes huge economic losses to the aquaculture industry. In this study, the three membrane proteins encoded by genes ORF25, ORF25C, and ORF25D of CyHV-2 were truncated and expressed in yeast, Pichia pastoris. Screening of the recombinant yeasts was done by detecting the truncated proteins using Western blot. Through immunogold labeling, it was shown that proteins binding the colloidal gold were presented on the surface of cells. In the experiment of inhibition of virus binding by the recombinant truncated proteins, the TCID50 of the tORF25 group (10(4.1)/ml) was lower than that of tORF25C (10(4.6)/ml) or tORF25D groups (10(5)/ml). These results suggested that the proteins may be involved in attachment of the virus to the cell surface. Healthy gibel carp were immunized with 20 μg of tORF25, tORF25C, and tORF25D proteins, and the control group received PBS. Interleukin 11 (IL-11) expression in the spleens of the immunized fish peaked at day 4 and the complement component C3 (C3) genes were significantly up-regulated at day 7 post-immunization. Specific antibodies were measured in the three immunized groups and the titer detected in the tORF25 group reached 327, that was significantly higher than the tORF25C (247) or tORF25D (228) groups. When the immunized fish were challenged with live CyHV-2 by intraperitoneal injection the relative percent survival (RPS) of the tORF25, tORF25C, and tORF25D immunized groups was 75%, 63%, and 54%, respectively. The feasibility of the P. pastoris yeast expression system for the production of the recombinant truncated proteins and their apparent bioactivity suggests that tORF25, tORF25C, and tORF25D are potential candidate vaccines against Cyprinid herpesvirus 2 infection in gibel carp.
authors.affiliation: College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430071, China; Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Jiang
authors.affiliation: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Ma
authors.affiliation: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors.forename: Yuding
authors.initials: Y
authors.lastname: Fan
authors.affiliation: College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430071, China.
authors.forename: Linlin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430071, China; Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China. Electronic address: zlb@yfi.ac.cn.
authors.forename: Lingbing
authors.initials: L
authors.lastname: Zeng
mesh_terms: D000818:Animals; D017756:Aquaculture; D004266:DNA Virus Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D004267:DNA Viruses / Q000276:immunology*; D005393:Fish Diseases / Q000276:immunology / Q000517:prevention & control*; D006054:Goldfish; D007113:Immunity, Innate; D010843:Pichia / Q000235:genetics; D017422:Sequence Analysis, DNA; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology*; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology*
chemical_list: D014763:Viral Matrix Proteins; D014765:Viral Vaccines
keywords: CyHV-2; Pichia pastoris expression; Protective immunity; Truncated expression