PlpE epitopes of Pasteurella multocida fusion protein as novel subunit vaccine candidates

Mostaan, Saied; Ghasemzadeh, Abbas; Ehsani, Parastoo; Sardari, Soroush; Shokrgozar, Mohammad Ali; Abolhassani, Mohsen; Nikbakht Brujeni, Gholamreza

PlpE epitopes of Pasteurella multocida fusion protein as novel subunit vaccine candidates

Authors

¹ Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran

² Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

³ Department of National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

⁴ Department of Immunology, Hybridoma Laboratory, Pasteur Institute of Iran, Tehran, Iran

⁵ Department of Microbiology and Immunology, University of Tehran, Tehran, Iran

Abstract

Background: Pasteurella multocida is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate. Materials and Methods: In this study, the fusion protein (PlpE1 + 2 + 3) and full PlpE genes (PlpE-Total) were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 Escherichia coli. Overlap polymerase chain reaction (PCR) using different primers for 5ˈ and 3ˈ end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning. Results: Cloning of both PlpE1 + 2 + 3 and PlpE-Total into the pET28a vector and their transform into the BL21 (DE3) E. coli host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration. Conclusion: Changing the vector to pBAD/gIII A and consequently changing the host to Top10 E. coli have resulted in sufficient expression, which shows that Top10 E. coli may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.

Keywords

References

1.	Peng Z, Wang X, Zhou R, Chen H, Wilson BA, Wu B. Pasteurella multocida: Genotypes and genomics. Microbiol Mol Biol Rev 2019;83:e00014-19.
2.	Wilson BA, Ho M. Pasteurella multocida: From zoonosis to cellular microbiology. Clin Microbiol Rev 2013;26:631-55.
3.	Dabo SM, Taylor JD and Confer AW. Pasteurella multocida and bovine respiratory disease. Animal Health Research Reviews 2007;8-2:129-150
4.	Ujvári B, Weiczner R, Deim Z, Terhes G, Urbán E, Tóth AR, et al. Characterization of Pasteurella multocida strains isolated from human infections. Comp Immunol Microbiol Infect Dis 2019;63:37-43.
5.	Farahani MF, Esmaelizad M, Jabbari AR. Investigation of iron uptake and virulence gene factors (fur, tonB, exbD, exbB, hgbA, hgbB1, hgbB2 and tbpA) among isolates of Pasteurella multocida from Iran. Iran J Microbiol 2019;11:191-7.
6.	Okerman L, Devriese LA. Failure of oil adjuvants to enhance immunity induced in mice by an inactivated rabbit Pasteurella multocida vaccine. Vaccine 1987;5:315-8.
7.	Dowling A, Hodgson JC, Dagleish MP, Eckersall PD, Sales J. Pathophysiological and immune cell responses in calves prior to and following lung challenge with formalin-killed Pasteurella multocida biotype A: 3 and protection studies involving subsequent homologous live challenge. Vet Immunol Immunopathol 2004;100:197-207.
8.	Hopkins BA, Huang TH, Olson LD. Differentiating turkey postvaccination isolants of Pasteurella multocida using arbitrarily primed polymerase chain reaction. Avian Dis 1998;42:265-74.
9.	Mariana S, Hirst R. The immunogenicity and pathogenicity of Pasteurella multocida isolated from poultry in Indonesia. Vet Microbiol 2000;72:27-36.
10.	Ahmad TA, Rammah SS, Sheweita SA, Haroun M, El-Sayed LH. Development of immunization trials against Pasteurella multocida. Vaccine 2014;32:909-17.
11.	Hansson M, Nygren PA, Ståhl S. Design and production of recombinant subunit vaccines. Biotechnol Appl Biochem 2000;32:95-107.
12.	Hatfaludi T, Al-Hasani K, Gong L, Boyce JD, Ford M, Wilkie IW, et al. Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE. PLoS One 2012;7:e39973.
13.	Singh AP, Singh S, Ranjan R, Gupta SK, Singh VP, Sharma B. Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2. J Vet Sci 2010;11:227-33.

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