Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies

Abstract Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of stonustoxin from Synanceia horrida and prepare antibodies against it. The SNTXα gene sequence was sourced from GenBank and codon-optimized to match the codon usage of E. coli BL21 (DE3). This optimized sequence was synthesized within the pET17b expression vector. IPTG induction triggered the expression of the SNTXα protein, which was subsequently purified using affinity chromatography. Following purification, the protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbits serum using a G protein column. The isolated antibodies were further confirmed using indirect ELISA. As a result of codon optimization, the Codon Adaptation Index (CAI) for the SNTXα cassette increased to 0.94. Predicted structures generated by the I-TASSER server exhibited good quality. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 12 mg/L. ELISA results demonstrated rabbits antibody titers detectable up to a 1:256,000 dilution. The isolated antibody from rabbits serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In conclusion, our study successfully expressed the primary toxic domain of stonustoxin in a prokaryotic host, enabling the production of rabbits antibodies for potential use in developing stonefish antivenom.