Regulation of porcine circovirus type 2-like particles expressed in baculovirus expression system

Abstract

Background

Porcine circovirus type 2 (PCV2) virus-like particles (VLPs) are an effective vaccine against post-weaning multisystemic wasting syndrome. Burst sequence (BS) and the chicken 5′-HS4 β-globin (HS4) insulator were used to improve the production of PCV2 VLPs in insect cells.

Results

BS was used to modify the polyhedron (polh) promoter, and the chicken HS4 insulator was inserted downstream of the target gene expression cassette. This enhanced effect was detected by EGFP, a generally used protein as reporter. The transcription-level analysis demonstrated that the double BS and the chicken HS4 insulator improved the transcription levels of the target genes. At the same time, the two regulatory elements also improved the PCV2 VLPs’ expression. PCV2 VLPs’ expression levels increased by 14 and 24 % at 72 h p.i. Further research observed an additive effect when the double BS and the chicken HS4 insulator simultaneously acted on the expression of PCV2 VLPs, with the PCV2 VLPs’ expression level increasing by 35 % at 72 h p.i.

Conclusions

BS and HS4 insulator can be used to improve the production of PCV2 VLPs in insect cells.

Keywords

Porcine circovirus type 2 / Virus-like particles / HS4 insulator / Burst sequence / Baculovirus infection / Recombinant protein production

References

[1] Blanchard P, Mahé D, Cariolet R, Keranflec’h A, Baudouard MA, Cordioli P, Albina E, Jestin A. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine, 2003, 21: 4565-4575.

[2] Chackerian B. Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines, 2007, 6: 381-390.

[3] Chae C. A review of porcine circovirus 2-associated syndromes and diseases. Vet J, 2005, 169: 326-336.

[4] Cox MMJ. Recombinant protein vaccines produced in insect cells. Vaccine, 2012, 30: 1759-1766.

[5] Crisci E, Bárcena J, Montoya M. Virus-like particles: the new frontier of vaccines for animal viral infections. Vet Immunol Immunopathol, 2012, 148: 211-225.

[6] Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, Haines D. Isolation of circovirus from lesions of pigs with post weaning multisystemic wasting syndrome. Can Vet J, 1998, 39: 44-51.

[7] Fan H, Ju C, Tong T, Huang H, Lv J, Chen H. Immunogenicity of empty capsids of porcine circovirus type 2 produced in insect cells. Vet Res Commun, 2007, 31: 487-496.

[8] Fernandes F, Teixeira AP, Carinhas N, Carrondo MJT, Alves PM. Insect cells as a production platform of complex virus-like particles. Expert Rev Vaccines, 2013, 12: 225-236.

[9] Grgacic EV, Anderson DA. Virus-like particles: passport to immune recognition. Methods, 2006, 40: 60-65.

[10] Harding JC. Post-weaning multisystemic wasting syndrome (PMWS): preliminary epidemiology and clinical presentation. Proc Am West Assoc Swine Pract, 1996, 28: 503.

[11] Harding JCS, Clark EG, Strokappe JH, Willson PI, Ellis JA. Postweaning multisystemic wasting syndrome: epidemiology and clinical presentation. J Swine Health Prod, 1998, 6: 249-254.

[12] Izumi M, Gilbert DM. Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts. J Cell Biochem, 1999, 76: 280-289.

[13] Kushnir N, Streatfield SJ, Yusibov V. Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development. Vaccine, 2012, 31: 58-83.

[14] Liu LJ, Suzuki T, Tsunemitsu H, Kataoka M, Ngata N, Takeda N, Wakita T, Miyamura T, Li TC. Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus. Arch Virol, 2008, 153: 2291-2295.

[15] Liu F, Ge S, Li L, Wu X, Liu Z, Wang Z. Virus-like particles: potential veterinary vaccine immunogens. Res Vet Sci, 2012, 93: 553-559.

[16] Liu F, Wu X, Li L, Liu Z, Wang Z. Use of baculovirus expression system for generation of virus-like particles: success and challenges. Protein Expr Purif, 2013, 90: 104-116.

[17] Liu Y, Zhang Y, Yao L, Hao H, Fu X, Yang Z, Du E. Enhanced production of porcine circovirus type 2(PCV2) virus-like particles in Sf9 cells by translational enhancers. Biotechnol Lett, 2015, 37: 1765-1771.

[18] López-vidal J, Gómez-Sebastián S, Bárcena J, Nuñez MC, Martínez-Alonso D, Dudognon B, Guijarro E, Escribano JM. Improved production efficiency of virus-like particles by the baculovirus expression vector system. PLoS One, 2015

[19] Manohar SL, Kanamasa S, Nishina T, Kato T, Park EY. Enhanced gene expression in insect cells and silkworm larva by modified polyhedrin promoter using repeated burst sequence and very late transcriptional factor-1. Biotechnol Bioeng, 2010, 107: 909-916.

[20] Nawagitgul P, Morozov I, Bolin SR, Harms PA, Sorden SD, Paul PS. Open reading frame 2 of porcine circovirus type2 encodes a major capsid protein. J Gen Virol, 2000, 81: 2281-2287.

[21] Noad R, Roy P. Virus-like particles as immunogens. Trends Microbiol, 2003, 11: 438-444.

[22] O’Reilly DR, Miller LK, Luckow VA. Titering virus stocks in baculovirus expression vectors: a laboratory manual, 1994, Oxford: Oxford University Press.

[23] Ooi BG, Rankin C, Miller LK. Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene. J Mol Biol, 1989, 210: 721-736.

[24] Pikaart MJ, Recillas-Targa F, Felsenfeld G. Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev, 1998, 12: 2852-2862.

[25] Pogranichniy RM, Yoon KJ, Yaeger M, Vaughn E, Stammer R, Roof M. Efficacy of experimental inactivated PCV2 vaccines for preventing PMWS in CDCD pigs. Proc Annu Meet Am Assoc Swine Vet, 2004, 35: 443-444.

[26] Rohrmann GF. Baculovirus molecular biology, 2011, Bethesda: United States National Library of Medicine.

[27] Sano KI, Maeda K, Maeda Y. Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence. FEBS Lett, 2002, 532: 143-146.

[28] Schiller JT, Castellsagué X, Garland SM. A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine, 2012, 30: 132-138.

[29] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc, 2008, 3: 1101-1108.

[30] Segalés J, Allan GM, Domingo M. Porcine circovirus diseases. Anim Health Res Rev, 2005, 6: 119-142.

[31] Tischer I, Gelderblom H, Vettermann W, Koch MA. A very small porcine virus with circular single-stranded DNA. Nature, 1982, 295: 64-66.

[32] Venkaiah B, Viswanathan P, Habib S, Hasnain S. An additional copy of the homologous region(hr1) sequence in the Autographa californica multinucleocapsid polyhedrosis virus genome promotes hyperexpression of foreign genes. Biochemistry, 2004, 43: 8143-8151.

[33] Wang X, Li L, Ding S, Huang X, Zhang J, Yin J, Zhong J. Chicken HS4 insulator significantly improves baculovirus-mediated foreign gene expression in insect cells by modifying the structure of neighbouring chromatin in virus minichromosome. J Biotechnol, 2009, 142: 193-199.

[34] Yamaji H. Suitability and perspectives on using recombinant insect cells for the production of virus-like particles. Appl Microbiol Biotechnol, 2014, 98: 1963-1970.

[35] Yin S, Sun S, Yang S, Shang Y, Cai X, Liu X. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli. Virol J, 2010, 7: 166.