Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E. coli

Subject Areas : Medical Microbiology

Abbas Doosti ¹

1 - Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran.

Received: 2012-02-20 Accepted : 2012-03-20 Published : 2013-01-01

Keywords: Cloning, Botulism, Neurotoxin, Heavy Chain,

Abstract :

Background and Objectives: Botulism is a food poisoning problem in which the transmission of neuromuscular stimuli is disrupted due to the activity of botulinum neurotoxins (BoNT) produced by Clostridium botulinum. The botulinum neurotoxins are the most known toxic substances that cause flaccid paralysis due to preventing the release of the neurotransmitter acetylcholine at neuromuscular junctions. The aim of this study was cloning the heavy chain (HC) domain of Clostridium botulinum neurotoxin gene in E. coli as a gene vaccine candidate. Material and Methods: In this study, the heavy chain of neurotoxin of Clostridium botulinum was fully amplified based on PCR and the specific primers. DNA fragment of heavy chain of neurotoxin gene was cloned by T/A cloning technique in PCR 8/GW/TOPO vector and the clone was transformed into E. coli. Results: Cloning of 2530 bp of neurotoxin was confirmed by PCR. The results of next step showed that the heavy chain of Clostridium botulinum neurotoxin was successfully cloned in       E. coli . Final confirmation of construct was done by BamHI and HindIII restriction enzymes. Conclusion: According to the results, the amplified gene can be used as a target for gene vaccines against botulism in the future.

References:

1. Dhaked RK, Singh MK, Singh P, Gupta P. Botulinum toxin: Bioweapon and magic drug. Indian J Med Res. 2010; 132: 489-503.

2. Shapiro RL, Hatheway C, Swerdlow DL.  Botulism in the United States: a clinical and epidemiologic review. Ann Intern Med.1998; 129: 221-228.

3. Tavakoli HR, Zeynali M, Mehrabi Tavana A. Scrutiny of food-borne botulism intoxication in Iran during 2003-2007 with the food hygiene view point. Hakim Res J. 2009; 11(4): 38- 46. [In Persian]

4. Rusnak JM, Smith LA. Botulinum neurotoxin vaccines past history and recent developments. Hum Vaccin. 2009; 5(12): 794-805.

5. Jain S, Ponmariappan S, Kumar O. Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein. Indian J Med Res. 2011; 134: 33-39.

6. Baldwin MR, Tepp WH, Przedpelski A, Pier CL, Bradshaw M, Johnson EA, Barbieri JT. Subunit vaccine against the seven serotypes of botulism. Infect. Immun. 2008; 76: 1314-1318.

7. Zichel R, Mimran A, Keren A, Barnea A, Steinberger-Levy I, Marcus D, Turgeman A, Reuveny S. Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E Produced in a cell-free expression system. Clin Vaccine Immunol. 2010; 17 (5): 784–792.

8. Macdonald TE, Helma CH, Shou Y, Valdez YE, Ticknor LO Foley BT, Davis SW, Hannett GE, Kelly-Cirino CD, Barash JR, Arnon SS, Lindström M, Korkeala H, Smith LA, Smith TJ, Hill KK. Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing. Appl Environ Microbiol. 2011; 77: 8625–8634.

9.Critchley EMR. A comparison of human and animal botulism. J R Soc Med. 1991; 84(5): 295-298.

10. Mereb G, Rosetti F, Castelli E, Ganuza R, Ottavioni, Cavallero D, Calderon A. Description of a botulism type C outbreak among waterbirds in the province of La Pamp, Argentina. Anaerobe. 1999; 5: 191-193.

11. Schoenbaum MA, Hall SM, Glock RD, Grant K, Jenny AL, Schiefer TJ, Sciglibaglio P, Whitlock RH. An outbreak of type C botulism in 12 horses and a mule. J Am Vet Med Assoc. 2000; 217(3): 365‑368.

12. Zhou Y, Foss S, Lindo P, Sarkar H, Singh BR. Hemagglutinin 33 of type A botulinum neurotoxin complex binds with synaptotagmin II. FEBS J. 2005; 272 (11): 2717- 26.

13. Bennett AM, Perkins SD, Holley JL. DNA vaccination protects against Botulinum neurotoxin type F. Vaccine. 2003; 21:3110–3117.

14. Jathoul AP, Holley JL, Garmory HS. Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters. Vaccine. 2004; 22:3942–3946.

15. Srivastava IK, Liu MA. Gene vaccines. Ann Intern Med. 2003; 138:550-559.

16. Yu Y, Zhang S, Sun Z, Wang S, Yu W. Enhanced immune responses using plasmid DNA replicon vaccine encoding the Hc domain of Clostridium botulinum neurotoxin serotype A. Vaccine. 2007; 25: 8843–8850.

17. Shyu RH, Shaio MF, Tang SS, Shyu HF, Lee CF, Tsai MH, Smith JE, Huang HH, Wey JJ, Huang JL, Chang HH. DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice. J Biomed Sci. 2000; 7:51–57.

18. Adams MR, Moss MD. Food Microbiology. Cambridge, 2nd ed, R.S.C 2002; 200-212.

19. Vahdani P. Botulism and food poisoning. Nazhat publication. 2001; 111- 117.

20. Vahdani P. The epidemiological survey on patients who were hospitalized in Logman Hakim Hospital with dignosis Botulism during 1997- 1998. The sixth congress in infectious diseases and tropical medicine. Shiraz University.

21. Porshafie A, Saadati M, Salimian J. Foodborne botulism due to consumption of local product cheese. Nabz J. 1996; 7: 44- 47. [In Persian]

22. Lalitha KV, Surendran PK. Occurrence of C. botulinum in fresh and cured fish in retail trade in Cochin (India). Int J Food Microbiol. 2002; 72: 169- 174.

23. Lalitha KV, Gopakamar K. Distribution and ecology of C. botulinum in fish and aquatic environments. J Food Microbiol. 2001; 17: 535- 41.

24. Sharpe AE, Brady CP, Byrne W, Moriarty J, O'Neill P, McLaughlin JG. Major outbreak of suspected botulism in a dairy herd in the Republic of Ireland. Vet Rec. 2008; 162 (13):409- 12.

25. Farrow BRH, Murrel WG, Stewart BJ, Zuber RM. Type C botulism in young gogs. Aust Vet J.1983; 60 (12): 374-377.

26- Liu MA. DNA vaccines: a review. J Int Med. 2003; 253: 402-410.