Original Article

Cloning and Expression of N-CFTX-1 Antigen from Chironex fleckeri in Escherichia coli and Determination of Immunogenicity in Mice


Background: Most jellyfish species are poisonous. Human victims of jellyfish sting each year are 120 million. Chironex fleckeri is a venomous box jellyfish that inflicts painful and potentially fatal stings to humans. The CfTX-1 is one of the antigenic proteins of venom that is suggested to stimulate the immune system for treatment and vaccine. This study aimed to clone and express the CfTX-1 antigen in E. coli and then to determine the synthesis of related antibody in the mice.

Methods: The study was performed in the Persian Gulf and Oman Sea Ecology Research Center, Bandar Abbas, Iran in autumn 2016. The synthetic CfTX-1 gene in PUC57 plasmid was purchased from Nedaye Fan Company. The 723 bp fragment of N-CfTX-1 was amplified by PCR, PUC57 plasmid containing CfTX-1 with BamHI SalI restriction enzyme sites were subcloned in pET28a [+] expression vector and transformed into E. coli BL21 (DE3). The CfTX-1 gene expression was induced by IPTG. Then antibody produced from the mice serum were isolated and confirmed by ELISA. After protein purification, resulted antigen was injected to mice in 4 repeats and then evaluated the rate of antibody in mice serum. Mice were challenged by the Carybdea alata.

Results: The 726 bp of N-CfTX-1 were cloned in a vector of expression pET28a [+] and confirmed by PCR, sequencing and enzymatic analysis. Moreover, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Then the antibody was isolated from mice serum and confirmed by ELISA test. The results showed that immunized mice tolerated 50x LD50[1] of jellyfish venom.

Conclusion: The CfTX-1 recombinant protein was able to protect the BALB/c mice against jellyfish venom. The produced protein can be used as a candidate for vaccine against jellyfish venom.

1. Jouiaei M, Yanagihara AA, Madio B, et al (2015). An-cient Venom Systems: A Review on Cnidaria Toxins. Toxins (Basel), 7(6): 2251–2271.
2. Jafari H, Honari H, Zargan J, Tamadoni Jahromi S (2019). Extraction the venom of Rhopilema no-madica from the Persian Gulf coast and the in-vestigation of its hemolytical activity. Yafte, 21 (3) :86-95.
3. Remigante A, Costa R, Morabito R, et al (2018). Im-pact of Scyphozoan Venoms on Human Health and Current First Aid Options for Stings. Toxins (Basel), 10(4) :133.
4. Brinkman D.L, Konstantakopoulos N, McInerney BV, et al (2014). Chironex fleckeri (Box Jellyfish) Venom Proteins: Expansion of a Cnidarian Tox-in Family that Elicits Variable Cytolytic and Cardi-ovascular Effects. J Biol Chem, 289 (8): 4798-812.
5. Diane L, Xinying J, Jeremy P, et al (2015). Transcrip-tome and venom proteome of the box jellyfish Chironex fleckeri . BMC Genomics, 16(1):407.
6. Brinkman D, Burnell J (2007). Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri. Toxicon, 50(6):850-60.
7. Danie M, Bollag S, Edelstein G (1991). Protein methods. 3rd ed, Chapters: 2, 3, 6, 8.
8. Bloom DA, Radwan FF, Burnett JW (2000). Tox-inological and immunological studies of capillary electrophoresis fractionated Chrysaora quinquecirrha (Desor) fishing tentacle and Chironex fleckeri South-cott nematocyst venoms. Comp Biochem Physiol CBP, 128(1):75-90.
9. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259):680-5.
10. Rusel D, Sambrook J (2001). Molecular cloning a laboratory Manual. 3rd Edition, New York, Cold Spring Harbor Laboratory Press,
11. Bradford M.M (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bind-ing. Anal Biochem, 72:248-54.
12. Salem HF, Fahmy A, Ali AMA (2011). Extended immunization of rats using microencapsulated cobra venom. British Journal of Pharmacology and Toxi-cology, 2(1): 43-50.
13. Andreosso A, Smout M J, Seymour J E (2014). Dose and time dependence of box jellyfish anti-venom. J Venom Anim Toxins Incl Trop Dis, 20:34.
14. Brinkman DL, Aziz A, Loukas, et al (2012). Venom Proteome of the Box Jellyfish Chironex fleckeri. PLoS One, 7(12) :e47866.
15. Brinkman DL, Burnell JN (2009). Biochemical and molecular characterization of cubozoan protein toxins. Toxicon, 54(8):1162-73.
16. Mariottini GL (2014). Hemolytic venoms from ma-rine cnidarian jellyfish – an overview. J Venom Res ,5: 22-32.
17. Brinkman D and Burncell J (2008). Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri. Toxicon, 51(5):853-63.
18. Isbister GK (2010). Antivenom efficacy or effective-ness: the Australian experience. Toxicology, 268(3):148-54.
19. Fan J, Zhuang Y, Li B (2013). Effects of collagen and collagen hydrolysate from jellyfish umbrella on histological and immunity changes of mice pho-toaging. Nutrients, 5(1): 223-33.
20. Winter KL, Isbister Gk, Jacoby T (2009). An in vivo comparison of the efficacy of CLS box jellyfish antivenom with antibodies raised against nemato-cyst-derived Chirnex fleckeri venom. Toxicol Lett, 187(2):94-8.
21. Sutherland SK (1992). Antivenom use in Australia Premedication, adverse reactions and the use of venom detection kits. Med J Aust, 157(11-12):734-9.
22. Alam MJ, Ashraf KUM (2013). Prediction of an Epitope-based Computational Vaccine Strategy for Gaining Concurrent Immunization against the Venom Proteins of Australian Box Jellyfish. Toxicol Int, 20(3): 235–253.
IssueVol 50 No 2 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijph.v50i2.5355
Chironex fleckeri Carybdea alata Venom

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How to Cite
JAFARI H, TAMADONI JAHROMI S, ZARGAN J, ZAMANI E, RANJBAR R, HONARI H. Cloning and Expression of N-CFTX-1 Antigen from Chironex fleckeri in Escherichia coli and Determination of Immunogenicity in Mice. Iran J Public Health. 50(2):376-383.