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.

Antibodies Produced Toward Recombinant RBD and Nucleocapsid Neutralize SARS-COV-2.

Background: The highly contagious SARS-COV-2 virus spread rapidly from China and formed a global pandemic. The virus has infected over 509 million people worldwide and killed about 6.32 million up to date. Up on invasion, the Receptor Binding Domain (RBD) of Spike protein plays a crucial role in the entry of the virus into the host cell. The virus N protein is another protein that has a critical role for genome packaging. Methods: As bioinformatics approaches, the cassette design, codon adaptation, and protein stability were investigated in this study. Synthetic genes of RBD and N were cloned separately in pET28a + expression vector. They were transferred into Escherichia coli (E. coli) BL21 DE3 host cell, and expression of recombinant proteins was induced with IPTG. The recombinant proteins were purified by column chromatography and approved by Western blotting. Animal immunization was performed with each of the recombinant proteins individually and in combination of the two. The antibody titer of the blood serum from control and immunized mice groups was determined by ELISA technique. Finally, the anti-spike neutralization test was performed. Results: The expression and purification of RBD protein were monitored on SDS-PAGE, two bands of about 28 and 45 kDa for RBD and N appeared on gel distinctly, which were further validated by Western blotting. According to ELISA results, related antibodies were traced to a dilution of 1/64000 in immunized sera. The neutralization test exhibited produced antibodies' potency to bind the virus proteins. Using SPSS software, statistical analysis was performed by Duncan's test and T-test. Conclusion: According to the present study, recombinant proteins, either RBD alone or in combination with N adequately stimulated the immune response, and the raised antibodies could neutralize the virus in in vitro test.