title: Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli.
abstract: Porcine circovirus type 2 (PCV2) capsid (Cap) protein has been successfully used as a vaccine to control porcine circovirus associated disease (PCVAD). Most PCV2 subunit vaccines are recombinant Cap protein expressed in baculovirus/insect cell expression system, but using this eukaryotic system is laborious and expensive. In our previous study, full-length of PCV2Cap protein expressed in Escherichia coli formed virus-like particles (VLPs). This expression system has the advantages of being relatively simple and inexpensive. In this study, we constructed a recombinant plasmid containing the full-length codon-optimized cap (ORF2) gene to improve high-level expression of recombinant Cap protein (rCap) with no changed amino acids. The highly water-soluble rCap protein was purified by a single-column, high-throughput fractionation procedure based on size exclusion chromatography. Yield was 10mg per 200ml bacterial culture. The rCap protein self-assembled into VLPs of diameter 25-30nm that contained exogenous nucleic acids. The immunogenicity of PCV2 VLPs was analyzed by immunizing mice. VLP-immunized mice mounted specific immune responses to PCV2. Thus, expression of rCap in E. coli was feasible for large-scale production of PCV2 VLPs, which could potentially be used for a VLP-based PCV2 vaccine.
authors.affiliation: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
authors.forename: Pei-Ching
authors.initials: PC
authors.lastname: Wu
authors.affiliation: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
authors.forename: Tzu-Yu
authors.initials: TY
authors.lastname: Chen
authors.affiliation: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
authors.forename: Jiun-Ni
authors.initials: JN
authors.lastname: Chi
authors.affiliation: Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC. Electronic address: mschien@dragon.nchu.edu.tw.
authors.forename: Maw-Sheng
authors.initials: MS
authors.lastname: Chien
authors.affiliation: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC. Electronic address: cjhuang@dragon.nchu.edu.tw.
authors.forename: Chienjin
authors.initials: C
authors.lastname: Huang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000917:Antibody Formation; D015153:Blotting, Western / Q000379:methods; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002850:Chromatography, Gel / Q000379:methods; D018173:Circoviridae Infections / Q000235:genetics / Q000276:immunology / Q000821:virology*; D017925:Circovirus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D013552:Swine; D013553:Swine Diseases / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D022223:Vaccines, Subunit; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D014771:Virion / Q000235:genetics
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; D058425:Vaccines, Virus-Like Particle
keywords: PCV2 capsid protein; Size exclusion chromatography; Vaccine; Virus-like particles