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Iran J Med Microbiol 2017, 11(3): 37-48 Back to browse issues page
Insilico Design and Construction of a Chimeric Gene Comprising the A Subunit of Vibrio cholera Pilin and the B Subunit of Cholera Toxin
Milad Amerian 1, Shahram Nazarian 2, Hosein Honari 1, Mohammad Ebrahim Minaie 1, Emad Kordbacheh 1
1- Biological Research Center, Faculty and Research Institute for Basic Sciences, Imam Hossein comprehensive University, Tehran, Iran
2- Biological Research Center, Faculty and Research Institute for Basic Sciences, Imam Hossein comprehensive University, Tehran, Iran , nazarian56@gmail.com
Abstract:   (2701 Views)

Background and Aims: Cholera is a lethal diarrheal disease that cause by Vibrio cholerae. Cholera toxin and colonization factor pili (tcpA) are the major virulence factor in V.cholerae pathogenesis. The B subunit of the enterotoxin )ctxB) which is responsible for toxin binding to eukaryotic cells and toxin-coregulated pili A (tcpA) that is essential for V.cholerae colonization, have immunogenic properties. Chimeric proteins carrying epitopes, linkers or adjuvant sequences could increase immunogenicity for recombinant antigens and can also elicit broad immune responses. The aim of this study was to design am immunogen against adherence and toxicity of V.cholorae.
Materials and Methods: ctxB and tcpA genes were analyzed for rare codons and gene optimization was performed using optimization software In 2017. The half-life and protein instability index was determined. Secondary and tertiary structure was predicted and evaluated. Linear and conformational epitopes were predicted. Recombinant pET28a/chimeric gene plasmid was transformed to E.coli BL21 DE3 and expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Results: Chimeric protein instability index was 24.04 and the codon adaptation index of chimeric constructs increased to 0.9. The tertiary predicted structure of the chimeric proteins confirmed and mRNA was stable. Conformational and linear epitopes were seen in both domains of the chimeric protein. Restriction analysis confirmed cloning of the gene into pET28a vector. Expression of recombinant protein in E.coli led to the production of chimeric protein with 37 k Da molecular weight.
Conclusions: According to the results of bioinformatics and recombinant protein expression, the design chimeric protein could be used as an immunogen to evaluate the immunity against cholera.

Keywords: Vibrio cholerae, Virulence factors, Cholera toxin, Bioinformatic desigen, Chimeric gene
Full-Text [PDF 1027 kb]   (814 Downloads)    
Type of Study: Original | Subject: Molecular Microbiology
1. Sanchez J, Holmgren J. Cholera toxin structure, gene regulation and pathophysiological and immunological aspects. Cellular and Molecular Life Sciences 2008;65(9):1347-60.
2. Mousavi SL, Nazarian S, Amani J, Karimi Rahgerdi A. Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA. Iranian biomedical journal 2008;12(1):15-21.
3. Mandal S, Mandal MD, Pal NK. Cholera: a great global concern. Asian Pacific journal of tropical medicine 2011;4(7):573-80.
4. Nazarian S, Arefpour M, Bagheripour M, Olad G. Bioinformatical Study and Evaluation of Expression of Cholera Toxin Subunit B Optimized Gene as a Vaccine Candidate. J Isfahan Med Sch 2014;32(279):378-87.
5. Mousavi SL, Rasooli I, Nazarian S, Amani J. Simultaneous detection of Escherichia coli O157: H7, toxigenic Vibrio cholerae, and Salmonella typhimurium by multiplex PCR. Archives of Clinical Infectious Diseases 2009;4(2):97-103.
6. Sánchez J, Holmgren J. Cholera toxin-a foe & a friend. Indian Journal of Medical Research 2011;133(2):153.
7. Bharati K, Ganguly NK. Cholera toxin: a paradigm of a multifunctional protein. Indian Journal of Medical Research 2011;133(2):179.
8. Faruque SM, Albert MJ, Mekalanos JJ. Epidemiology, Genetics, and Ecology of ToxigenicVibrio cholerae. Microbiology and molecular biology reviews 1998;62(4):1301-14.
9. Banerjee R, Das B, Nair GB, Basak S. Dynamics in genome evolution of Vibrio cholerae. Infection, Genetics and Evolution 2014;23:32-41.
10. Baldauf KJ, Royal JM, Hamorsky KT, Matoba N. Cholera toxin B: one subunit with many pharmaceutical applications. Toxins 2015;7(3):974-96.
11. Taylor RK, Miller VL, Furlong DB, Mekalanos JJ. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proceedings of the National Academy of Sciences 1987;84(9):2833-7.
12. Viel T, Dransart E, Nemati F, Henry E, Thézé B, Decaudin D, et al. In vivo tumor targeting by the B-subunit of shiga toxin. Molecular imaging 2008;7(6):7290.2008. 00022.
13. Johansson E-L, Bergquist C, Edebo A, Johansson C, Svennerholm A-M. Comparison of different routes of vaccination for eliciting antibody responses in the human stomach. Vaccine 2004;22(8):984-90.
14. Kundu J, Mazumder R, Srivastava R, Srivastava BS. Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge. FEMS Immunology & Medical Microbiology 2009;56(2):179-84.
15. Childers BM, Klose KE. Regulation of virulence in Vibrio cholerae: the ToxR regulon. Future Microbiology 2007; 2(3):335-44.
16. Odumosu O, Nicholas D, Yano H, Langridge W. AB toxins: a paradigm switch from deadly to desirable. Toxins 2010;2(7):1612-45.
17. Abraham E, Wunderink R, Silverman H, Perl TM, Nasraway S, Levy H, et al. Efficacy and safety of monoclonal antibody to human tumor necrosis factor α in patients with sepsis syndrome: a randomized, controlled, double-blind, multicenter clinical trial. Jama 1995;273(12):934-41.
18. Shen My, Sali A. Statistical potential for assessment and prediction of protein structures. Protein science 2006;15(11):2507-24.
19. Nazarian S, Gargari SLM, Rasooli I, Amani J, Bagheri S, Alerasool M. An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. Journal of microbiological methods 2012;90(1):36-45.
20. Puigbo P, Guzman E, Romeu A, Garcia-Vallve S. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic acids research 2007;35(suppl 2):W126-W31.
21. Doytchinova IA, Flower DR. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC bioinformatics 2007;8(1):4.
22. Patwary NIA, Islam MS, Sohel M, Ara I, Sikder MOF, Shahik SM. In silico structure analysis and epitope prediction of E3 CR1-beta protein of Human Adenovirus E for vaccine design. Biomedical Journal 2016;39(6):382-90.
23. Garg VK, Avashthi H, Tiwari A, Pramod PAJ. MFPPI–Multi FASTA ProtParam Interface. Bioinformation 2016;12(2):74-7.
24. Chakraborty C, Agrawal A. Computational analysis of C-reactive protein for assessment of molecular dynamics and interaction properties. Cell biochemistry and biophysics 2013;67(2):645-56.
25. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic acids research 2003;31(13):3406-15.
26. Barrick JE, Roberts RW. Sequence analysis of an artificial family of RNA‐binding peptides. Protein Science 2002;11(11):2688-96.
27. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nature methods 2015;12(1):7-8.
28. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic acids research 2007;35(suppl 2):W407-W10.
29. Ansari HR, Raghava GP. Identification of conformational B-cell Epitopes in an antigen from its primary sequence. Immunome research 2010;6(1):6.
30. Amani J, Salmanian AH, Rafati S, Mousavi SL. Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157: H7. Vaccine 2010;28(42):6923-9.
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Amerian M, Nazarian S, Honari H, Minaie M E, kordbacheh E. Insilico Design and Construction of a Chimeric Gene Comprising the A Subunit of Vibrio cholera Pilin and the B Subunit of Cholera Toxin. Iran J Med Microbiol. 2017; 11 (3) :37-48
URL: http://ijmm.ir/article-1-665-en.html

year 11, Issue 3 (July - August 2017) Back to browse issues page
مجله میکروب شناسی پزشکی ایران Iranian Journal of Medical Microbiology
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