Expression of Soluble c-CfTXB Protein from Chironex fleckeri Jellyfish Fused to SUMO Tag in Escherichia coli BL21 (DE3) and Evaluation of Immunogenicity in Syrian Mice

Mehdi  Bakhshian; Hossein  Honari; Hossein Samiei  Abianeh; Seyed Mojtaba  Aghaie

doi:10.18502/ssu.v34i4.22071

Mehdi Bakhshian Department of Biology, School of Basic Sciences, Imam Hossein comprehensive university, Tehran, Iran.
Hossein Honari Department of Biology, School of Basic Sciences, Imam Hossein comprehensive university, Tehran, Iran.
Hossein Samiei Abianeh Department of Medical Biotechnology and Nanotechnology, School of Medicine, University of Medical Sciences Mashhad, Iran.
Seyed Mojtaba Aghaie Department of Biology, School of Basic Sciences, Imam Hossein comprehensive university, Tehran, Iran

DOI: https://doi.org/10.18502/ssu.v34i4.22071

Keywords: CfTXB, Chironex fleckeri, Jellyfish, Soluble protein, SUMO

Abstract

Introduction: The box jellyfish Chironex fleckeri is a toxin-producing species whose cytotoxic and neurotoxic effects have been well demonstrated in living organisms. Due to the presence of various bioactive proteins within its toxin, acute exposure can lead to temporary paralysis or even death under severe conditions. This study aimed to solubilize and evaluate the immunogenicity of the engineered SUMO-tagged recombinant c-CfTXB protein as an antigen, by comparing its immune response to the untagged form in animal model.

Methods: The StXB gene fragment was excised from the pET-28aSUMO-c-CfTXB-StXB vector using enzymatic digestion. Subsequently, the recombinant proteins SUMO-c-CfTXB and c-CfTXB were expressed in competent prokaryotic hosts following IPTG induction, and purified through Ni-NTA affinity chromatography. Protein identity was confirmed by Western blot analysis. Following immunization of mouse groups, antibody titers were measured using indirect ELISA, and protective efficacy was evaluated through a toxin challenge. Data analysis was performed using One-way ANOVA combined with survival analysis.

Results: Successful protein expression and separation were confirmed by SDS-PAGE and Western blot analysis, demonstrating that the SUMO tag enhanced the solubility of the target protein. In the toxin challenge test, only the vaccinated groups survived (100%) due to a significant increase in antibody titers (Pvalue c-CfTXB›0.0001 and Pvalue SUMO-c-CfTXB›0.0003).

Conclusion: Fusion of the SUMO tag to the engineered c-CfTXB protein improved its folding, expression yield, purification efficiency, and solubility. These findings suggest that the successful immunogenicity of the antigen in the mouse model may pave the way for the design of recombinant vaccines to neutralize box jellyfish venom.