Please wait a minute...
Journal of Integrative Agriculture  2017, Vol. 16 Issue (07): 1601-1608    DOI: 10.1016/S2095-3119(16)61605-2
Animal Science · Veterinary Science Advanced Online Publication | Current Issue | Archive | Adv Search |
The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles
YANG Cui1, 2, 3*, XU Yu1, 2, 3*, JIA Ren-yong1, 2, 3*, LIU Si-yang1, 2, 3*, WANG Ming-shu1, 2, 3, ZHU De-kang1, 3, CHEN Shun1, 2, 3, LIU Ma-feng1, 2, 3, ZHAO Xin-xin1, 2, 3, SUN Kun-feng1, 2, 3, JING Bo3, YIN Zhong-qiong3, CHENG An-chun1, 2, 3
1 Avian Disease Research Centre, Sichuan Agricultural University, Chengdu 611130, P.R.China
2 Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, P.R.China
3 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
Abstract      The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus’s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCV Cap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pastoris GS115. The strains that displayed the phenotype of Mut+ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What’s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15–20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the results demonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.
Keywords:  capsid gene        codon-optimization        duck circovirus        virus-like particles  
Received: 08 November 2016   Accepted:
Fund: 

This study was supported by the National Science and Technology Support Program (2015BAD12B05), the China Agricultural Research System (CARS-43-8), the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province, China (2014NZ0030), the Ministry of Education Program of China (20125103110013), the Sichuan Province Research Programs, China (2013HH0042/2013TD0015/2014-002).

Corresponding Authors:  Correspondence JIA Ren-yong, Tel/Fax: +86-28-86291176, E-mail: jiary@sicau.edu.cn    
About author:  YANG Cui, E-mail: yc_itzel@163.com;

Cite this article: 

YANG Cui, XU Yu, JIA Ren-yong, LIU Si-yang, WANG Ming-shu, ZHU De-kang, CHEN Shun, LIU Ma-feng, ZHAO Xin-xin, SUN Kun-feng, JING Bo, YIN Zhong-qiong, CHENG An-chun . 2017. The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles. Journal of Integrative Agriculture, 16(07): 1601-1608.

Allnutt F T, Bowers R M, Rowe C G, Vakharia V N, LaPatra S E, Dhar A K. 2007. Antigenicity of infectious pancreatic necrosis virus VP2 subviral particles expressed in yeast. Vaccine, 25, 4880–4888.

Bachmann M F, Rohrer U H, Kündig T M, Bürki K, Hengartner H, Zinkernagel R M. 1993. The influence of antigen organization on B cell responsiveness. Science, 262, 1448–1451.

Banda A, Galloway-Haskins R I, Sandhu T S, Schat K A. 2007. Genetic analysis of a duck circovirus detected in commercial Pekin ducks in New York. Avian Diseases, 51, 90–95.

Brun A, Bárcena J, Blanco E, Borrego B, Dory D, Escribano J M, Gall-Reculé G L, Ortego J, Dixon L K. 2011. Current strategies for subunit and genetic viral veterinary vaccine development. Virus Research, 157, 1–12.

Bucarey S A, Noriega J, Reyes P, Tapia C, Sáenz L, Zuñiga A, Tobar J A. 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.

Cereghino J L, Cregg J M. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24, 45–66.

Cha S Y, Kang M, Cho J G, Jang H K. 2013. Genetic analysis of duck circovirus in Pekin ducks from South Korea. Poultry Science, 92, 2886–2891.

Chen C L, Wang P X, Lee M S, Shien J H, Shieh H K, Ou S J, Chen C H, Chang P C. 2006. Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus. Avian Diseases, 50, 92–95.

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. Journal of Virology, 77, 4928–4937.

Cregg J M, Cereghino J L, Shi J, Higgins D R. 2000. Recombinant protein expression in Pichia pastoris. Molecular Biotechnology, 16, 23–52.

Ding J, Zhang C, Gao M, Hou G, Liang K, Li C, Ni J, Li Z, Shi Z. 2014. Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy. Journal of Biotechnology, 177, 35–44.

Farnós O, Fernández E, Chiong M, Parra F, Joglar M, Méndez L, Rodríguez E, Moya G, Rodríguez D, Lleonart R. 2009. Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: Advantages for immunization strategies and vaccine implementation. Antiviral Research, 81, 25–36.

Fringuelli E, Scott A, Beckett A, McKillen J, Smyth J, Palya V, Glavits R, Ivanics E, Mankertz A, Franciosini M. 2005. Diagnosis of duck circovirus infections by conventional and real-time polymerase chain reaction tests. Avian Pathology, 34, 495–500.

Guo L J, Lu Y H, Huang L P, Wei Y W, Liu C M. 2011. Identification of a new antigen epitope in the nuclear localization signal region of porcine circovirus type 2 capsid protein. Intervirology, 54, 156–163.

Hattermann K, Schmitt C, Soike D, Mankertz A. 2003. Cloning and sequencing of duck circovirus (DuCV). Archives of Virology, 148, 2471–2480.

Heath L, Williamson A L, Rybicki E P. 2006. The capsid protein of beak and feather disease virus binds to the viral dna and is responsible for transporting the replication-associated protein into the nucleus. Journal of Virology, 80, 7219–7225.

Kwag H L, Kim H J, Chang D Y, Kim H J. 2012. The production and immunogenicity of human papillomavirus type 58 virus-like particles produced in Saccharomyces cerevisiae. Journal of Microbiology, 50, 813–820.

Lewis G D, Metcalf T G. 1988. Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Applied and Environmental Microbiology, 54, 1983–1988.

Liu Q, Willson P, Attoh-Poku S, Babiuk L A. 2001. Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein. Protein Expression and Purification, 21, 115–120.

Liu S N, Zhang X X, Zou J F, Xie Z J, Zhu Y L, Qin Z, Zhou E M, Jiang S J. 2010. Development of an indirect ELISA for the detection of duck circovirus infection in duck flocks. Veterinary Microbiology, 145, 41–46.

Li Z, Wang X, Zhang R, Chen J, Xia L, Lin S, Xie Z, Jiang S. 2014. Evidence of possible vertical transmission of duck circovirus. Veterinary Microbiology, 174, 229–232.

Lu Y Y, Jia R Y, Zhang Z L, Wang M S, Xu Y, Zhu D K, Chen S, Liu M F, Yin Z Q, Chen X Y, Cheng A C. 2014. In vitro expression and development of indirect ELISA for capsid protein of duck circovirus without nuclear localization signal. International Journal of Clinical & Experimental Pathology, 7, 4938–4944.

Matczuk A K, Krawiec M, Wieliczko A. 2015. A new duck circovirus sequence, detected in velvet scoter (Melanitta fusca) supports great diversity among this species of virus. Virology Journal, 12, 1–7.

Misinzo G, Meerts P, Bublot M, Mast J, Weingartl H, Nauwynck H. 2005. Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31. Journal of General Virology, 86, 2057–2068.

Paliard X, Liu Y, Wagner R, Wolf H, Baenziger J, Walker C M. 2000. Priming of strong, broad, and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T cells after administration of a virus-like particle vaccine in rhesus macaques. AIDS Research and Human Retroviruses, 16, 273–282.

Plummer E M, Manchester M. 2011. Viral nanoparticles and virus-like particles: Platforms for contemporary vaccine design. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 3, 174–196.

Pumpens P, Grens E. 2001. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology, 44, 98–114.

Rao N H, Babu P B, Rajendra L, Sriraman R, Pang Y Y S, Schiller J T, Srinivasan V. 2011. Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles. Vaccine, 29, 7326–7334.

Soike D, Albrecht K, Hattermann K, Schmitt C, Mankertz A. 2004. Novel circovirus in mulard ducks with developmental and feathering disorders. Veterinary Record, 154, 792–793.

Stewart M, Bonne N, Shearer P, Sharp M, Raidal S. 2007. Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination. Journal of Virological Methods, 141, 181–187.

Tegerstedt K, Franzén A V, Andreasson K, Joneberg J, Heidari S, Ramqvist T, Dalianis T. 2005. Murine polyomavirus virus-like particles (VLPs) as vectors for gene and immune therapy and vaccines against viral infections and cancer. Anticancer Research, 25, 2601–2608.

Tu Y, Wang Y, Wang G, Wu J, Liu Y, Wang S, Jiang C, Cai X. 2013. High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris. Applied Microbiology and Biotechnology, 97, 2867–2875.

Walia R, Dardari R, Chaiyakul M, Czub M. 2014. Porcine circovirus-2 capsid protein induces cell death in PK15 cells. Virology, 468–470, 126–132.

Wang D, Xie X, Zhang D, Ma G, Wang X, Zhang D. 2011. Detection of duck circovirus in China: A proposal on genotype classification. Veterinary Microbiology, 147, 410–415.

Xia M, Farkas T, Jiang X. 2007. Norovirus capsid protein expressed in yeast forms virus-like particles and stimulates systemic and mucosal immunity in mice following an oral administration of raw yeast extracts. Journal of Medical Virology, 79, 74–83.

Xiang Q W. 2012. Study of biological charateristics of ORF2 and ORF3 of DuCV. MSc thesis, Shandong Agricultural University, China. pp. 63–69. (in Chinese)

Xiang Q W, Wang X, Xie Z J, Sun Y N, Zhu Y L, Wang S J, Liu H J, Jiang S J. 2012. ORF3 of duck circovirus: A novel protein with apoptotic activity. Veterinary Microbiology, 159, 251–256.

Xiang Q W, Zou J F, Wang X, Sun Y N, Gao J M, Xie Z J, Wang Y, Zhu Y L, Jiang S J. 2013. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus. Virology, 436, 112–117.

Yadava A, Ockenhouse C F. 2003. Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infection and Immunity, 71, 4961–4969.

Yan Y. 2009. Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus Type 2. Molecular Immunology, 46, 327–334.

Zahn K. 1996. Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth. Journal of Bacteriology, 178, 2926–2933.

Zhang X X, Liu S N, Xie Z J, Kong Y B, Jiang S J. 2012. Complete genome sequence analysis of duck circovirus strains from Cherry Valley duck. Virologica Sinica, 27, 154–164.

Zhang Z, Jia R, Wang M, Lu Y, Zhu D, Chen S, Yin Z, Wang Y, Chen X, Cheng A. 2013. Complete genome sequence of the novel duck circovirus strain GH01 from southwestern China. Genome Announcements, 1, e00166-12.

Zhou J Y, Shang S B, Gong H, Chen Q X, Wu J X, Shen H G, Chen T F, Guo J Q. 2005. In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal. Journal of Biotechnology, 118, 201–211.
No related articles found!
No Suggested Reading articles found!