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:: Volume 28, Issue 3 (Scientific Journal of Kurdistan University of Medical Sciences 2023) ::
SJKU 2023, 28(3): 13-23 Back to browse issues page
Expression, Purification and evaluation of the Immunogenicity of RecombinantC-terminus of the Receptor-Binding Domain of Neurotoxin Botulinum Protein Type B (BoNT/B-HcC)
Hossein Samiei Abianeh1 , Shahram Nazarian2 , Mohammad ebrahim Minaei3 , Jafar Amani4 , Amir Sajjad Hojjati razgi5
1- Ph.D. Candidate of Medical Biotechnology, Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2- Associate Professor, Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran. , nazarian@ihu.ac.ir.
3- Associate Professor, Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran
4- Professor, Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
5- MSc student, Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.
Abstract:   (2787 Views)
Background and Aim: Botulism, a syndrome caused by food poisoning, results from use of food contaminated with the botulinum toxin, which is very dangerous and deadly. Botulism is caused by the effects of bacterial toxins on the terminals of the motor nerves. Botulinum neurotoxins are among the most potent toxins. The aim of this study was to investigate expression, purification and, evaluation of the immunogenicity of the recombinant BoNT/B-HcC protein as an immunogen candidate in mice.
Materials and Methods: The C-terminus of the receptor-binding domain of  botulinum neurotoxin type B(BoNT/B-HcC) was selected as the antigen for bioinformatics evaluations. The pET17b-BoNT/B-HcC plasmid was transferred to E. coli BL21(DE3) by heat shock. The recombinant protein was purified and analyzed by SDS-PAGE. After verification of the recombinant protein by western blot, immunization of mice was performed. Antibody titers of recombinant proteins were evaluated by indirect ELISA and the results were compared using t-test.
Results: The codon adaptation index (CAI) of the optimized gene was 0.99. The percentage of codons having high-frequency distribution was improved to 78%. Restriction analysis confirmed the 1119 bp construct gene and the recombinant protein with a molecular weight of 43.8 kDa was expressed in the prokaryotic host. The total yield of purified protein was 23 mg of protein per liter of culture. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer was significantly different compared to that of the control sample.
Conclusion: The designed recombinant antigen showed high antigenicity that could be considered as an immunogen against botulinum type B neurotoxin in future studies.


 
Keywords: Clostridium botulinum, Vaccine, ‌ Immunizations, Antibody, Catalytic domain
Full-Text [PDF 778 kb]   (543 Downloads)    
Type of Study: Original Research | Subject: Biotechnology
Received: 2022/04/12 | Accepted: 2022/07/6 | Published: 2023/07/31
References
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23. Van Ermengem E. A new anaerobic bacillus and its relation to botulism. Rev Infect Dis. 1979:701-19. [DOI:10.1093/clinids/1.4.701]
24. Pickett A. On the discovery of Bacillus botulinus. The Botulinum Journal. 2008;1(1):5-6. [DOI:10.1504/TBJ.2008.018957]
25. Johnson EA, Bradshaw M. Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective. Toxicon. 2001;39(11):1703-22. [DOI:10.1016/S0041-0101(01)00157-X] [PMID]
26. Valipour E, Moosavi ML, Amani J, Nazarian S. High level expression, purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E. World J Microbiol Biotechnol. 2014;30(6), 1861-1867. [DOI:10.1007/s11274-014-1609-0] [PMID]
27. Pourshafie M, Saifie M, Shafiee A, Vahdani P, Aslani M, Salemian J. An outbreak of food-borne botulism associated with contaminated locally-made cheese in Iran. Scand J Infect Dis. 1998;30(1):92-4. [DOI:10.1080/003655498750002385] [PMID]
28. Baghban R, Gargari SL, Rajabibazl M, Nazarian S, Bakherad H. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris. Biotechnol Appl Biochem. 2016;63(2):200-5. [DOI:10.1002/bab.1226] [PMID]
29. Hatheway CL. Clostridium botulinum and other clostridia that produce botulinum neurotoxin. In Clostridium botulinum. 2018; 2-20. [DOI:10.1201/9781315139623-1]
30. Goonetilleke A, Harris J. Clostridial neurotoxins. J Neurol, Neurosurg Psychiatry. 2004;75. [DOI:10.1136/jnnp.2004.046102] [PMID] []
31. Dolly J, Aoki K. The structure and mode of action of different botulinum toxins. Eur J Paediatr Neurol. 2006;13:1-9. [DOI:10.1111/j.1468-1331.2006.01648.x] [PMID]
32. Heymann DL. Control of communicable diseases manual. Am J Public Health. 2008;19. [DOI:10.1071/NB08016] [PMID]
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34. Ehrt S, Schnappinger D. Isolation of plasmids from E. coli by alkaline lysis. E coli Plasmid Vectors. Springer; 2003. p. 75-8. [DOI:10.1385/1-59259-409-3:75] [PMID]
35. Fairweather NF, Lyness VA, Maskell DJ. Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli. Infect Immun. 1987;55(11):2541-5. [DOI:10.1128/iai.55.11.2541-2545.1987] [PMID] []
36. Smith LA. Development of recombinant vaccines for botulinum neurotoxin. Toxicon. 1998;36(11):1539-48. [DOI:10.1016/S0041-0101(98)00146-9] [PMID]
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39. Dolimbek BZ, Steward LE, Aoki KR, Atassi MZ. Immune recognition of botulinum neurotoxin B: antibody-binding regions on the heavy chain of the toxin. Mol Immunol. 2008;45(4):910-24. [DOI:10.1016/j.molimm.2007.08.007] [PMID]
40. Rostami H, Moosavi SJ, Ebrahimi F, Hajizadeh A. Cloning and Expression of the Catalytic Domain of Botulinum Neurotoxin Type E in E. coli. J. Mazandaran Univ Med Sci. 2013;22(97):148-57.
41. Baradaran M, Ebrahimi F, Nazarian S, Hajizade A, Tarverdizadeh Y. Subcloning and Assessment of the Expression of Synthetic Botulinum Neurotoxin Type B Binding Domain (BD/B). J Qom Univ Med Sci. 2016;10(5), 29-37.
42. Sharma S, Zhou Y, Singh BR. Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A. Protein Expression Purif. 2006;45(2):288-95. [DOI:10.1016/j.pep.2005.07.020] [PMID]
43. Clayton MA, Clayton JM, Brown DR, Middlebrook JL. Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli. Infect Immun. 1995;63(7):2738-42. [DOI:10.1128/iai.63.7.2738-2742.1995] [PMID] []
44. Zhou Y, Singh BR. Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain. Protein Expression Purif. 2004;34(1):8-16. [DOI:10.1016/j.pep.2003.10.015] [PMID]
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Samiei Abianeh H, Nazarian S, Minaei M E, Amani J, Hojjati razgi A S. Expression, Purification and evaluation of the Immunogenicity of RecombinantC-terminus of the Receptor-Binding Domain of Neurotoxin Botulinum Protein Type B (BoNT/B-HcC). SJKU 2023; 28 (3) :13-23
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Volume 28, Issue 3 (Scientific Journal of Kurdistan University of Medical Sciences 2023) Back to browse issues page
مجله علمی دانشگاه علوم پزشکی کردستان Scientific Journal of Kurdistan University of Medical Sciences
مجله علمی دانشگاه علوم پزشکی کردستان Scientific Journal of Kurdistan University of Medical Sciences
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