High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris

Appl Microbiol Biotechnol. 2013 Apr;97(7):2867-75. doi: 10.1007/s00253-012-4540-z. Epub 2012 Nov 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Viral / blood
  • Antigens, Viral / biosynthesis*
  • Antigens, Viral / genetics
  • Antigens, Viral / immunology*
  • Capsid Proteins / biosynthesis*
  • Capsid Proteins / genetics
  • Capsid Proteins / immunology*
  • Circovirus / genetics*
  • Circovirus / immunology
  • Circovirus / isolation & purification
  • Codon
  • Gene Expression*
  • Genetic Vectors
  • Molecular Sequence Data
  • Pichia / genetics*
  • Plasmids
  • Promoter Regions, Genetic
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Sequence Analysis, DNA
  • Swine
  • Viral Vaccines / administration & dosage
  • Viral Vaccines / immunology

Substances

  • Antibodies, Viral
  • Antigens, Viral
  • Capsid Proteins
  • Codon
  • Recombinant Proteins
  • Viral Vaccines

Associated data

  • GENBANK/JX867373