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

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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.

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References

  • Allan GM, Meehan B, Todd D, Kennedy S, McNeilly F, Ellis J, Clark EG, Harding JC, Espuna E, Botner A, Charreyre C (1998) Novel porcine circoviruses from pigs with wasting disease syndromes. Vet Rec 142:467–468

    Article  CAS  Google Scholar 

  • Allan GM, McNeilly E, Kennedy S, Meehan B, Moffett D, Malone F, Ellis J, Krakowka S (2000) PCV-2-associated PDNS in Northern Ireland in 1990. Porcine dermatitis and nephropathy syndrome. Vet Rec 146:711–712

    CAS  Google Scholar 

  • Allan GM, Krakowka S, Ellis J (2002) PCV2: ticking time bomb? http://prod.epi.pigprogress.net/PageFiles/26871/001_boerderij-download-PP5468D01.pdf

  • Bretthauer RK, Castellino FJ (1999) Glycosylation of Pichia pastoris derived proteins. Biotechnol Appl Biochem 30:193–200

    CAS  Google Scholar 

  • Brunel L, Neugnot V, Landucci L, Boze WN, Moulin G, Bigey F (2004) High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris. J Biotechnol 111:41–50

    Article  CAS  Google Scholar 

  • Bucarey SA, Noriega J, Reyes P, Tapia C, Sáenz L, Zuñiga A, Tobar JA (2009) The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine 27:5781–5790

    Article  CAS  Google Scholar 

  • Cai L, Ni J, Xia Y, Zi Z, Ning K, Qiu P, Li X, Wang B, Liu Q, Hu D, Yu X, Zhou Z, Zhai X, Han X, Tian K (2012) Identification of an emerging recombinant cluster in porcine circovirus type 2. Virus Res 165:95–102

    Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  CAS  Google Scholar 

  • Cheng CC, Lee YF, Lin NN, Wu CL, Tung KC, Chiu YT (2011) Bronchiolitis obliterans organizing pneumonia in Swine associated with porcine circovirus type 2 infection. J Biomed Biotechnol 2011. doi:10.1155/2011/245728

  • Cid-Arregui A, Juárez V, zur Hausen H (2003) A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of the protein in mammalian cells and is useful for DNA immunization studies. J Virol 77:4928–4937

    Article  CAS  Google Scholar 

  • Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T (2009) Expression in the yeast Pichia pastoris. Methods Enzymol 463:169–189

    Article  CAS  Google Scholar 

  • Fachinger V, Bischoff R, Jedidia SB, Saalmuller A, Elbers K (2008) The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex. Vaccine 26:1488–1499

    Article  CAS  Google Scholar 

  • Fan H, Ju C, Tong T, Huang H, Lv J, Chen H (2007) Immunogenicity of empty capsids of porcine circovius type 2 produced in insect cells. Vet Res Commun 31:487–496

    Article  CAS  Google Scholar 

  • Farnós O, Fernández E, Chiong M, Parra F, Joglar M, Méndez L, Rodríguez E, Moya G, Rodríguez D, Lleonart R, González EM, Alonso A, Alfonso P, Suárez M, Rodríguez MP, Toledo JR (2009) Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: advantages for immunization strategies and vaccine implementation. Antivir Res 81:25–36

    Article  Google Scholar 

  • Fort M, Olvera A, Sibila M, Segales J, Mateu E (2007) Detection of neutralizing antibodies in postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs. Vet Microbiol 125:244–255

    Article  CAS  Google Scholar 

  • Fort M, Sibila M, Pérez-Martín E, Nofrarías M, Mateu E, Segalés J (2009) One dose of a porcine circovirus 2 (PCV2) sub-unit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model. Vaccine 27:4031–4037

    Article  CAS  Google Scholar 

  • Grau-Roma L, Segalés J (2007) Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky’s disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain. Vet Microbiol 119:144–151

    Article  CAS  Google Scholar 

  • Guo L, Lu Y, Wei Y, Huang L, Wu H, Liu C (2011) Porcine circovirus genotype 2a (PCV2a) and genotype 2b (PCV2b) recombinant mutants showed significantly enhanced viral replication and altered antigenicity in vitro. Virology 419:57–63

    Article  CAS  Google Scholar 

  • Hamel AL, Lin LL, Nayar GP (1998) Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 72:5262–5267

    CAS  Google Scholar 

  • Harding JC (2007) Porcine circovirus diseases (PCVD): the brutal facts. In: American Association of Swine Veterinarians-Proceedings Conference, Orlando, FL, 5 March, pp 349–352

  • Harding JC, Clark EG (1997) Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS). http://www.aasv.org/shap/issues/v5n5/v5n5p201.pdf

  • Horlen KP, Dritz SS, Nietfeld JC, Henry SC, Hesse RA, Oberst R, Hays M, Anderson J, Rowland RRR (2008) A field evaluation of mortality rate and growth performance in pigs vaccinated against porcine circovirus type 2. J Am Vet Med Assoc 232:906–912. http://avmajournals.avma.org/doi/full/10.2460/javma.232.6.906

    Google Scholar 

  • Huang H, Yang P, Luo H, Tang H, Shao N, Yuan T, Wang Y, Bai Y, Yao B (2008) High-level expression of a truncated 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation. Appl Microbiol Biotechnol 78:95–103

    Article  CAS  Google Scholar 

  • Ju C, Fan H, Tan Y, Liu Z, Xi X, Cao S, Wu B, Chen H (2005) Immunogenicity of a recombinant pseudorabies virus expressing ORF1-ORF2 fusion protein of porcine circovirus type 2. Vet Microbiol 109:179–190

    Article  CAS  Google Scholar 

  • Kanekiyo M, Matsuo K, Hamatake M, Hamano T, Ohsu T, Matsumoto S, Yamada T, Yamazaki S, Hasegawa A, Yamamoto N, Honda M (2005) Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag. J Virol 8:8716–8723

    Article  Google Scholar 

  • Kim J, Chae C (2004) Expression of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 in porcine circovirus 2-induced granulomatous inflammation. J Comp Pathol 131:121–126

    Article  CAS  Google Scholar 

  • Kim J, Chung HK, Chae C (2003) Association of porcine circovirus 2 with porcine respiratory disease complex. Vet J 166:251–256

    Article  CAS  Google Scholar 

  • Kixmoller M, Ritzmann M, Eddicks M, Saalmuller A, Elbers K, Fachinger V (2008) Reduction of PMWS-associated clinical signs and co-infections by vaccination against PCV2. Vaccine 26:3443–3451

    Article  Google Scholar 

  • Li Y, Zhang B, Chen X, Chen Y, Cao Y (2010) Improvement of Aspergillus sulphureus endo-beta-1,4-xylanase expression in Pichia pastoris by codon optimization and analysis of the enzymic characterization. Appl Biochem Biotechnol 160:1321–1331

    Article  CAS  Google Scholar 

  • Liu Q, Tikoo SK, Babiuk LA (2001a) Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2. Virology 285:91–99

    Article  CAS  Google Scholar 

  • Liu Q, Willson P, Attoh-Poku S, Babiuk LA (2001b) Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein. Protein Expres Purif 21:115–120

    Article  CAS  Google Scholar 

  • Mankertz A, Mueller B, Steinfeldt T, Schmitt C, Finsterbusch T (2003) New reporter gene-based replication assay reveals exchangeability of replication factors of porcine circovirus types 1 and 2. J Virol 77:9885–9893

    Article  CAS  Google Scholar 

  • Martelli P, Ferrari L, Morganti M, De Angelis E, Bonilauri P, Guazzetti S, Caleffi A, Borghetti P (2011) One dose of a porcine circovirus 2 subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus- associated disease under field conditions. Vet Microbiol 149:339–351

    Article  CAS  Google Scholar 

  • Mateusen B, Maes DG, Van Soom A, Lefebvre D, Nauwynck HJ (2007) Effect of a porcine circovirus type 2 infection on embryos during early pregnancy. Theriogenology 68:896–901

    Article  CAS  Google Scholar 

  • Menéndez C, Martínez D, Trujillo LE, Mazola Y, González E, Pérez ER, Hernández L (2012) Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris. Appl Microbiol Biotechnol. doi:10.1007/s00253-012-4270-2

  • Nawagitgul P, Morozov I, Bolin SR, Harms PA, Sorden SD, Paul PS (2000) Open reading frame 2 of porcine circovirus type 2 encodes amajor capsid protein. J Gen Virol 81:2281–2287

    CAS  Google Scholar 

  • Nawagitgul P, Harms PA, Morozov I, Thacker BJ, Sorden SD, Lekcharoensuk C, Paul PS (2002) Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV. Clin Diagn Lab Immunol 9:33–40

    CAS  Google Scholar 

  • O’Connor B, Gauvreau H, West K, Bogdan J, Ayroud M, Clark EG, Konoby C, Allan G, Ellis JA (2001) Multiple porcine circovirus 2-associated abortions and reproductive failure in a multisite swine production unit. Can Vet J 42:551–553

    Google Scholar 

  • Opriessnig T, Meng XJ, Halbur PG (2007) Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest 19:591–615

    Article  Google Scholar 

  • Outchkourov NS, Stiekema WJ, Jongsma MA (2002) Optimization of the expression of equistatin in Pichia pastoris. Protein Expr Purif 24:18–24

    Article  CAS  Google Scholar 

  • Segales J, Olvera A, Grau-Roma L, Charreyre C, Nauwynck H, Larsen L, Dupont K, McCullough K, Ellis J, Krakowka S, Mankertz A, Fredholm M, Fossum C, Timmusk S, Stockhofe-Zurwieden N, Beattie V, Armstrong D, Grassland B, Baekbo P, Allan G (2008) PCV-2 genotype definition and nomenclature. Vet Rec 162:867–868

    Article  CAS  Google Scholar 

  • Sigoillot M, Brockhoff A, Lescop E, Poirier N, Meyerhof W, Briand L (2012) Optimization of the production of gurmarin, a sweet-taste-suppressing protein, secreted by the methylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol. doi:10.1007/s00253-012-3897-3

  • Sinclair G, Choy FY (2002) Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 26:96–105

    Article  CAS  Google Scholar 

  • Szeredi L, Szentirmai C (2008) Proliferative and necrotising pneumonia and severe vascular lesions in pigs naturally infected with porcine circovirus type 2. Acta Vet Hung 56:101–109

    Article  Google Scholar 

  • Teng D, Fan Y, Yang YL, Tian ZG, Luo J, Wang JH (2007) Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Appl Microbiol Biotechnol 74:1074–1083

    Article  CAS  Google Scholar 

  • Trible BR, Rowland RR (2012) Genetic variation of porcine circovirus type 2 (PCV2) and its relevance to vaccination, pathogenesis and diagnosis. Virus Res 164:68–77

    Article  CAS  Google Scholar 

  • Wang F, Guo X, Ge X, Wang Z, Chen Y, Cha Z, Yang H (2009) Genetic variation analysis of Chinese strains of porcine circovirus type 2. Virus Res 145:151–156

    Article  CAS  Google Scholar 

  • Wellenberg GJ, Stockhofe-Zurwieden N, de Jong MF, Boersma WJ, Elbers AR (2004) Excessive porcine circovirus type 2 antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case–control study. Vet Microbiol 99:203–214

    Article  CAS  Google Scholar 

  • Wellenberg GJ, Bouwkamp FT, Wolf PJ, Swart WA, Mombarg MJ, de Gee AL (2010) A study on the severity and relevance of porcine circovirus type 2 infections in Dutch fattening pigs with respiratory diseases. Vet Microbiol 142:217–224

    Article  CAS  Google Scholar 

  • Woo JH, Liu YY, Mathias A, Stavrou S, Wang Z, Thompson J, Neville DM (2002) Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris. Protein Expr Purif 25:270–282

    Article  CAS  Google Scholar 

  • Yadava A, Ockenhouse CF (2003) Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun 71:4961–4969

    Article  CAS  Google Scholar 

  • Zhou JY, Shang SB, Gong H, Chen QX, Wu JX, Shen HG, Chen TF, Guo JQ (2005) In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal. J Biotechnol 118:201–211

    Article  CAS  Google Scholar 

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Acknowledgments

This study is supported by 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), and Fundamental Research Funds for Central Public Welfare Research Institutes (2012ZL079).

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Correspondence to Xuehui Cai.

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Tu, Y., Wang, Y., Wang, G. et al. High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris . Appl Microbiol Biotechnol 97, 2867–2875 (2013). https://doi.org/10.1007/s00253-012-4540-z

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