Immunogenicity of N-CfTX-2 Antigen of Chironex fleckeri Jellyfish in Mice

Abstract

Background and Objectives: Chironex. fleckeri is the most toxic and dangerous marine species. CfTX-2 is one of the antigenic proteins of jellyfish venom that has been suggested for stimulation of the immune system, for treatment, and as a candidate for vaccine. The purpose of this study was designing, optimization, synthesis, and expression of the N-CfTX-2 gene in E. coli and investigation of the production of antibodies and immunization in mice.   Methods: In this experimental study, the complete sequence of N-CfTX-2 gene was obtained from NCBI (with accession number AFQ00677.1). After optimizing the codon of E. coli host, other bioinformatics studies needed for better gene expression were performed, such as protein stability and mRNA structure. The gene sequence was ordered for synthesis and replication in the pET21a (+) vector. The recombinant vector was transformed into E. coli BL21 and protein expression was induced by IPTG. After purification of the protein, the obtained antigen was injected into the mice in four steps and the amount of antibody produced in their serum, was measured. Then, the produced antibody was isolated from the serum and confirmed by ELISA assay. Sixty days after the first injection, the mice were challenged by the Chironex fleckeri jellyfish venom.   Results: The N-CfTX-2 gene sequence cloned in the expression vector pET21a (+) was confirmed by PCR, and the produced recombinant protein was confirmed by SDS-PAGE and Western blot. Moreover, the immunized mice tolerated jellyfish venom 50 x LD50.   Given the antigenic properties of N-CfTX-2 and antibody production in laboratory animal, this protein can be used as a vaccine candidate in future studies.   &nbsp