Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus

Yong Zhou; Yuding Fan; Scott E LaPatra; Jie Ma; Jin Xu; Yan Meng; Nan Jiang; Lingbing Zeng

doi:10.1016/j.vaccine.2015.08.054

Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus

Vaccine. 2015 Oct 13;33(42):5662-5669. doi: 10.1016/j.vaccine.2015.08.054. Epub 2015 Aug 22.

Authors

Yong Zhou¹, Yuding Fan², Scott E LaPatra³, Jie Ma², Jin Xu², Yan Meng², Nan Jiang², Lingbing Zeng⁴

Affiliations

¹ Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China; College of Fisheries, Huazhong Agricultural University, Wuhan 430071, China.
² Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
³ Research Division, Clear Springs Foods, Inc., P.O. Box 712, Buhl, ID 83316, USA.
⁴ Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China. Electronic address: zlb@yfi.ac.cn.

PMID: 26303874
DOI: 10.1016/j.vaccine.2015.08.054

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.

Keywords: Chinese giant salamander; Iridovirus; MCP; Pichia pastoris expression; Protective immunity.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Antibodies, Viral
Base Sequence
Capsid Proteins / genetics
Capsid Proteins / immunology*
Cloning, Molecular
DNA Virus Infections / prevention & control
DNA Virus Infections / veterinary*
Iridovirus*
Molecular Sequence Data
Neutralization Tests
Pichia / metabolism
Urodela / virology*
Vaccines, Virus-Like Particle / immunology
Viral Vaccines / immunology*

Substances

Antibodies, Viral
Capsid Proteins
Vaccines, Virus-Like Particle
Viral Vaccines
major capsid protein, Iridovirus