Majallah-i Dānishgāh-i ’Ulūm-i Pizishkī-i Īlām (Dec 2019)
Subcloning and Expression of the Chimeric Antigen C-CFTX1-STxB of the Jellyfish Venom and its Antigenicity Assessment in Syrian Mice
Abstract
Introduction: Box jellyfish stings are painful and may be life-threatening. The venom of Chironex fleckeri contains a variety of bioactive proteins as well as two of the most abundant proteins, namely CfTX-1 and CfTX-2 which cannot be isolated easily using electrophoresis or chromatography techniques. Recombinant expression technology may offer an alternative to the isolation of native C.fleckeri venom protein. This study aimed at expressing C-CfTX1-STxB protein in Escherichia coli and assessing its antigenicity in Syrian mice. Materials & Methods: Synthesis of the artificial CfTX1complete gene was prepared in plasmid pUC57. The C-cftx1 was cloned using a polymerase chain reaction (PCR) and subcloned with BamHI and SalI restriction enzyme sites in pET28a-stxB expression vector and transformed into E.coli. Gene expression was artificially induced by Isopropyl β- d-1-thiogalactopyranoside. After the purification of the protein and its injection into the Syrian mice, the amount of produced antibody was measured in the serum. The rats were also challenged by the venom of the jellyfish (i.e., Rhopilema nomadic). Findings: In this experimental study, the C-CfTX1-STxB gene was cloned in the expression vector pET28a (+), sequenced by PCR, and analyzed by enzymatic analysis. Moreover, the produced recombinant protein was confirmed by Western blotting. The produced antibody in the serum was quantified using an enzyme-linked immunosorbent assay. Discussion & Conclusions: After 60 days, the immunized mice tolerated 50x LD50 of jellyfish venom. Considering the ineffectiveness of cardiotoxicity and neurotoxicity of the recombinant protein, this produced protein can be suggested as a jellyfish venom vaccine candidate in Syrian mice or at a later stage of a clinical trial in humans.
