title: High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris.
abstract: 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 α-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 μg/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.
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Yabin
authors.initials: Y
authors.lastname: Tu
authors.forename: Yanqun
authors.initials: Y
authors.lastname: Wang
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Jianan
authors.initials: J
authors.lastname: Wu
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Liu
authors.forename: Shujie
authors.initials: S
authors.lastname: Wang
authors.forename: Chenggang
authors.initials: C
authors.lastname: Jiang
authors.forename: Xuehui
authors.initials: X
authors.lastname: Cai
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D017925:Circovirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003062:Codon; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D013552:Swine; D014765:Viral Vaccines / Q000008:administration & dosage / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D003062:Codon; D011994:Recombinant Proteins; D014765:Viral Vaccines