Abstract
BackgroundPorcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC).MethodsThe Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared.ResultsELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge.ConclusionA novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.
Highlights
Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide
We explored the feasibility of ELPylation technology for simple purification and immunogenicity improvement of PCV2 virus-like particle (VLP)
Vector construction and protein expression To enhance the immunogenicity of PCV2b Cap protein, the coding sequence was fused with the coding sequences for the virus neutralizing (VN) epitopes of PCV2a, 2d and 2e
Summary
Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Vaccination is an effective and economical method for the control of PCV2 infection. At least five commercial PCV2 vaccines are available in the international market, including the inactivated vaccine, capsid protein-based vaccines and chimerical PCV1/2 vaccine [3]. The inactivated vaccine is produced commonly in PK-15 cells with low yield due to poor propagation of PCV2 in PK-15 cells [4]. The capsid protein-based virus-like particle (VLP) vaccine can be produced in E. coli or baculovirus system, which requires expensive ultracentrifugation or chromatography for purification [5, 6]. Reduction of PCV2 vaccine production cost is a key priority for veterinary research
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