title: Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus.
abstract: The major capsid protein (MCP) is the main immunogenic protein of iridoviruses, that has been widely used as an immunogen in vaccination trials. In this study, the codon-optimized giant salamander iridovirus (GSIV) MCP gene (O-MCP) was synthesized and cloned into a pPICZα B vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The expression of the O-MCP protein was detected by the Bradford protein assay, SDS-PAGE, Western blotting and electron microscopy. The Bradford protein assay indicated that the concentration of the O-MCP expressed was about 40 μg/ml in culture supernatants. SDS-PAGE analysis revealed that the O-MCP had a molecular weight of about 66 kDa and reacted with a His-specific MAb that was confirmed by Western blotting. Electron microscopy observations revealed that the purified O-MCP could self-assemble into virus-like particles. Healthy giant salamanders were vaccinated by intramuscular injection with the O-MCP antigen at a dose of 20 μg/individual. The numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly at day 3 and reached a peak at day 5 post-immunization. Meanwhile, the differential leukocyte counts of monocytes and neutrophils increased significantly at day 5 post-immunization compared to that of the control group. The percentage of lymphocytes was 71.33 ± 3.57% at day 21 post-immunization. The neutralization assay showed that the serum neutralizing antibody titer reached 321 at day 21 post-immunization. The GSIV challenge test revealed that the relative percent survival of Chinese giant salamanders vaccinated with O-MCP was 78%. These results indicated that the O-MCP antigen expressed by the Pichia pastoris system elicited significant immune response in the Chinese giant salamander against GSIV and might represent a potential yeast-derived vaccine candidate that could be used for the control of disease caused by the giant salamander iridovirus.
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China; College of Fisheries, Huazhong Agricultural University, Wuhan 430071, China.
authors: Yong
authors: Y
authors: Zhou
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors: Yuding
authors: Y
authors: Fan
authors: Research Division, Clear Springs Foods, Inc., P.O. Box 712, Buhl, ID 83316, USA.
authors: Scott E
authors: SE
authors: LaPatra
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors: Jie
authors: J
authors: Ma
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors: Jin
authors: J
authors: Xu
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors: Yan
authors: Y
authors: Meng
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
authors: Nan
authors: N
authors: Jiang
authors: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China. Electronic address: zlb@yfi.ac.cn.
authors: Lingbing
authors: L
authors: Zeng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004266:DNA Virus Infections / Q000517:prevention & control / Q000662:veterinary*; D017994:Iridovirus; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010843:Pichia / Q000378:metabolism; D014562:Caudata / Q000821:virology*; D058425:Vaccines, Virus-Like Particle / Q000276:immunology; D014765:Viral Vaccines / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D058425:Vaccines, Virus-Like Particle; D014765:Viral Vaccines; C081132:major capsid protein, Iridovirus
keywords: Chinese giant salamander; Iridovirus; MCP; Pichia pastoris expression; Protective immunity