期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 97, 期 7, 页码 2867-2875出版社
SPRINGER
DOI: 10.1007/s00253-012-4540-z
关键词
Porcine circovirus type 2; Capsid protein; Pichia pastoris; Expression; Immunogenicity
资金
- Natural Science Foundation of Heilongjiang Province of China [C201047]
- National High Technology Research and Development Program of China [2011AA10A213]
- Key Technology R&D Program of Harbin [2010AA6AN083]
- Excellent Youth Foundation of Heilongjiang Province of China [JC201020]
- Fundamental Research Funds for Central Public Welfare Research Institutes [2012ZL079]
The porcine circovirus type 2 (PCV2) capsid protein (Cap) is an important antigen for the development of vaccines. To achieve high-level expression of recombinant PCV2 Cap in Pichia pastoris, the wild-type Cap (wt-Cap) and optimized Cap (opti-Cap) gene fragments encoding the same amino acid sequence of PCV2 were amplified by PCR using DNA from lymph nodes of postweaning multisystemic wasting syndrome-suffered pigs and synthesized based on the codon bias of the methylotrophic yeast P. pastoris, respectively. The wt-Cap and opti-Cap gene fragments were inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the alcohol oxidase 1 (AOX1) promoter and alpha-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmids, designated as pPIC9K-wt-Cap and pPIC9K-opti-Cap, were linearized using SacI and transformed into P. pastoris GS115 by electroporation. The expressed intracellular soluble opti-Cap reached 174 mu g/mL without concentration in a shake flask and kept good reactivity to PCV2-specific positive sera, whereas the wt-Cap could not be detectable throughout three times electroporation. Strong specific PCV2-Cap antibodies were elicited from piglets immunized with vaccine based on opti-Cap. To the best of our knowledge, the achieved opti-Cap yield is the highest ever reported. Our results demonstrated that codon optimization play an important role on the high-level expression of a codon-optimized PCV2-Cap gene in P. pastoris, and the vaccine based on opti-Cap may be a potential subunit vaccine candidate.
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