Heterologous synthesis of N and M fragments of capsid protein VP2 of avian infectious bursal disease virus in yeast Pichia pastoris

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Abstract

BACKGROUND: Infectious bursal disease is one of the most common and economically important viral diseases of birds. Vaccination is currently the most effective way to control IBD. Subunit vaccines contain only the immunogenic protein of the pathogen or its fragments, but do not contain other proteins, lipopolysaccharides, toxins, which avoids vaccination side effects.

AIM: The aim of the work was to obtain yeast Pichia pastoris strains that synthesize and secrete the fragments of major coat protein VP2 of the infectious bursal disease virus.

MATERIALS AND METHODS: The DNA sequences encoding the N and M fragments of VP2 protein, were cloned under the control of the AOX1 gene promoter and integrated into the genome of P. pastoris strains X-33 (mut+) and GS115 (his4).

RESULTS: The analysis of proteins secreted by the obtained strains revealed the presence of additional proteins with a molecular weights corresponding to the target proteins.

CONCLUSIONS: Thus, the obtained strains of P. pastoris – producers of N and M fragments of VP2 protein can be used for antigen production to create a subunit vaccine against avian IBD.

About the authors

Andrey M. Rumyantsev

Saint Petersburg State University

Email: rumyantsev-am@mail.ru
ORCID iD: 0000-0002-1744-3890
SPIN-code: 9335-1184
Scopus Author ID: 55370658800

Cand. Sci. (Biol.), Senior Researcher

Russian Federation, Saint Petersburg

Mikhail A. Tsygankov

Saint Petersburg State University

Email: mial.tsygankov@yandex.ru
ORCID iD: 0000-0002-2513-6655
SPIN-code: 1098-0995
Scopus Author ID: 56252740000
ResearcherId: H-4691-2013

Research engineer

Russian Federation, Saint Petersburg

Vladislav V. Veretennikov

Saint Petersburg State University

Email: vlad.veretennikov.96@mail.ru
SPIN-code: 3412-1396
Scopus Author ID: 57219380560

Junior Researcher

Russian Federation, Saint Petersburg

Elena V. Sambuk

Saint Petersburg State University

Email: esambuk@mail.ru
ORCID iD: 0000-0003-0837-0498
SPIN-code: 8281-8020
Scopus Author ID: 6603061322
ResearcherId: H-6895-2013

Dr. Sci. (Biol.), Professor

Russian Federation, Saint Petersburg

Marina V. Padkina

Saint Petersburg State University

Author for correspondence.
Email: mpadkina@mail.ru
ORCID iD: 0000-0002-4051-4837
SPIN-code: 7709-0449
Scopus Author ID: 57200427270

Dr. Sci. (Biol.), Professor

Russian Federation, Saint Petersburg

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Supplementary files

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2. Figure 1. Schemes of the resulting plasmids. (a) Location of nucleotide and amino acid sequences corresponding to the M (208–351 a.u.) and N (18–139 a.m.) fragments of the VP2 protein of the IBD virus; b — scheme of plasmids pPICZαB/(625-1053) and pPICZαB/(52-417). Bacterial origin of replication (pUC origin), zeocin resistance gene [coding sequence — Zeo(R), bacterial promoter — EM7, yeast promoter — TEF1, transcription terminator — CYC1], expression cassette [alcohol oxidase gene promoter 1 (AOX1 promoter)] ], the sequence of the signal peptide (alpha-factor), the sequences encoding fragments of the VP2 protein of the IBD virus (M-VP2 and N-VP2), the sequences encoding the c-myc epitope and the histidine tag (6xHis), the transcription terminator of the alcohol oxidase 1 gene ( AOX1 transcription terminator)

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3. Figure 2. Electropherograms of PCR products. a — PCR results: 1 — with primers to the N fragment sequence and pPICZαВ/(52-417) plasmid as a template, 3 — with primers to the M fragment sequence and pPICZαВ/(625-1053) plasmid; b — PCR results: with primers for the N fragment sequence and genomic DNA of strain N-X-33 (4), strain N-GS115 (5); with primers to the sequence of fragment M and genomic DNA of strain M-X-33 (6); strain M-GS115 (7). Tracks 2 and 8 — DNA Ladder 100+ bp marker (Evrogen, Russia)

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4. Figure 3. Electropherogram of proteins of the culture medium of strain M-X-33 (a) and the results of Western blot hybridization with antibodies to the c-myc epitope (b). 1 - Culture liquid of the original strain X-33; 2 and 4, PageRuler Prestained Protein Ladder markers (Thermofisher Scientific, USA); 3 — culture liquid of strain M-X-33 synthesizing secretory fragment M VP2 (208–351 aa) with c-myc epitope and histidine label. The arrow indicates the band corresponding to the secretory fragment M of the VP2 protein.

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5. Figure 4. Electrophoregram of proteins purified from the culture liquid of yeast strains that produce the N and M fragments of the VP2 protein (a) and the results of Western blot hybridization with antibodies to the c-myc epitope (b). 1 - Proteins of the culture fluid of the original strain X-33 (negative control); 2 — secretory fragment N VP2 (18–139 aa.) synthesized by strain N-X-33; 3 and 4 — M VP2 secretory fragment (208–351 aa.) synthesized by strains M-X-33 and M-GS115, respectively; 5 — PageRuler Prestained Protein Ladder marker (Thermofisher Scientific, USA). The bands corresponding to the synthesized proteins are indicated (white arrow) and the bands presumably corresponding to the dimer of the VP2 M-fragment (black arrow)

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6. Figure 5. Results of Western blot hybridization with antibodies to the c-myc epitope. Purified VP2 protein M fragment (208–351 aa) in PBS buffer with 250 mM imidazole without boiling (1); with boiling (2); purified M fragment of VP2 protein in PBS buffer without boiling (4); with boiling (5); 3, 6 — PageRuler Prestained Protein Ladder marker (Thermofisher Scientific, USA). Bands corresponding to synthesized proteins (white arrow) and bands presumably corresponding to the dimer of the VP2 M-fragment (black arrow) are indicated. Separately, high-molecular protein aggregates (Ag) are indicated.

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