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

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 optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s 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 the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.

Bioinformatics design of recombinant chimeric protein containing SipD and LptD immunogens and evaluation of its immunogenicity against Salmonella Typhimurium.

The growing emergence of resistant bacteria is the current global concern for the humans and animals. Vaccination could be the desirable method to preventing such infectious diseases. Safe and effective vaccines are urgently needed to manage and prevent Salmonella contamination. Subunit vaccines are safe approaches for the protection against Salmonella spp. The bioinformatics methods were performed to determine the gene structure. Gene cassette (rLPSI) was ordered in pET28a (+), and cloned into E.coli BL21 (DE3), and the recombinant protein was expressed using IPTG (1 mM). Mice were immunized by subcutaneous administration of recombinant protein. Then, the mice were challenged by oral administration of 100LD50 of live S. Typhimurium. The Codon adaptation index of the chimeric gene was multiplied by 0.92. Validation results showed that >90% of residues lie in the desired or extra allowed area of the Ramachandran plot. The recombinant protein (65.9 kDa) was expressed in E.coli. Antibody titers in vaccinated mice were significantly different from those in the control groups. Recombinant protein immunization of the mice provided 90% and 70% protection against 10LD50 and 100LD50 of S. Typhimurium, respectively. In general, the results showed the high efficiency of rLPSI chimeric protein as a protective antigen against S. Typhimurium infection. The rLPSI chimeric protein could be an effective recombinant vaccine candidate against S. Typhimurium infection, but more research is needed.