Bioinformatic Analysis and Recombinant Expression of the Stonustoxin β-Subunit for Polyclonal Antibody Development.
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| Formato: | Artículo científico |
| Lenguaje: | en |
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Iranian journal of immunology : IJI
2025
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| _version_ | 1868266146706227200 |
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| author | Rahmani, Mohammadreza Nazarian, Shahram Samiei-Abianeh, Hossein Aghaie, Seyed Mojtaba Sadeghi, Davoud |
| author_facet | Rahmani, Mohammadreza Nazarian, Shahram Samiei-Abianeh, Hossein Aghaie, Seyed Mojtaba Sadeghi, Davoud Rahmani, Mohammadreza Nazarian, Shahram Samiei-Abianeh, Hossein Aghaie, Seyed Mojtaba Sadeghi, Davoud |
| collection | PubMed - marine biology |
| contents | Bioinformatic Analysis and Recombinant Expression of the Stonustoxin β-Subunit for Polyclonal Antibody Development. Rahmani, Mohammadreza Nazarian, Shahram Samiei-Abianeh, Hossein Aghaie, Seyed Mojtaba Sadeghi, Davoud Animals Rabbits Recombinant Proteins Computational Biology Escherichia coli Antibodies, Neutralizing Epitope Mapping Protein Subunits Gene Expression Stonefish (Synanceia spp.) are among the most venomous marine organisms. Their venom contains stonustoxin (SNTX), a heterodimeric toxin that induces severe hemolytic and myotoxic effects primarily mediated by its β-subunit. To produce a recombinant SNTX β-subunit and develop neutralizing polyclonal antibodies against SNTX. A DNA cassette encoding immunogenic regions of the β-sntx was designed using bioinformatics analysis, and codon-optimized for expression in E. coli. The construct was cloned into pET17b vector, and expressed in E. coli BL21 (DE3). The recombinant protein was purified via Ni-NTA affinity chromatography. For antibody production, rabbits were immunized subcutaneously with the recombinant protein emulsified in Freund's complete adjuvant, followed by booster doses at 2-week intervals. Antiserum was purified using protein G chromatography, and antibody titers were assessed by indirect Enzyme-Linked Immunosorbent Assay (ELISA). Epitope mapping revealed key immunogenic regions within residues 124-654 of the β-SNTX subunit. Following codon optimization, the codon adaptation index (CAI) increased to 0.93. Recombinant protein production was confirmed by SDS-PAGE and Western blot demonstrating successful purification of a 73 kDa recombinant protein (including TRX/His-tags), with a yield of 40 mg/L. Immunization of rabbits elicited a strong polyclonal IgG response, with antibody titers reaching 1:25,600 following the third booster. Purified IgG (1.8 mg/mL) exhibited high sensitivity in ELISA, detecting the recombinant β-SNTX at concentrations as low as 31.25 ng. The recombinant β-SNTX subunit demonstrated strong immunogenicity, inducing high-affinity antibodies with specific binding activity against the native toxin. The resulting antiserum demonstrated significant neutralization potential, highlighting its promise for antivenom development. |
| format | Artículo científico |
| id | pubmed_41025585 |
| institution | PubMed |
| language | en |
| publishDate | 2025 |
| publisher | Iranian journal of immunology : IJI |
| record_format | pubmed |
| spellingShingle | Bioinformatic Analysis and Recombinant Expression of the Stonustoxin β-Subunit for Polyclonal Antibody Development. Rahmani, Mohammadreza Nazarian, Shahram Samiei-Abianeh, Hossein Aghaie, Seyed Mojtaba Sadeghi, Davoud Animals Rabbits Recombinant Proteins Computational Biology Escherichia coli Antibodies, Neutralizing Epitope Mapping Protein Subunits Gene Expression Bioinformatic Analysis and Recombinant Expression of the Stonustoxin β-Subunit for Polyclonal Antibody Development. Rahmani, Mohammadreza Nazarian, Shahram Samiei-Abianeh, Hossein Aghaie, Seyed Mojtaba Sadeghi, Davoud Animals Rabbits Recombinant Proteins Computational Biology Escherichia coli Antibodies, Neutralizing Epitope Mapping Protein Subunits Gene Expression Stonefish (Synanceia spp.) are among the most venomous marine organisms. Their venom contains stonustoxin (SNTX), a heterodimeric toxin that induces severe hemolytic and myotoxic effects primarily mediated by its β-subunit. To produce a recombinant SNTX β-subunit and develop neutralizing polyclonal antibodies against SNTX. A DNA cassette encoding immunogenic regions of the β-sntx was designed using bioinformatics analysis, and codon-optimized for expression in E. coli. The construct was cloned into pET17b vector, and expressed in E. coli BL21 (DE3). The recombinant protein was purified via Ni-NTA affinity chromatography. For antibody production, rabbits were immunized subcutaneously with the recombinant protein emulsified in Freund's complete adjuvant, followed by booster doses at 2-week intervals. Antiserum was purified using protein G chromatography, and antibody titers were assessed by indirect Enzyme-Linked Immunosorbent Assay (ELISA). Epitope mapping revealed key immunogenic regions within residues 124-654 of the β-SNTX subunit. Following codon optimization, the codon adaptation index (CAI) increased to 0.93. Recombinant protein production was confirmed by SDS-PAGE and Western blot demonstrating successful purification of a 73 kDa recombinant protein (including TRX/His-tags), with a yield of 40 mg/L. Immunization of rabbits elicited a strong polyclonal IgG response, with antibody titers reaching 1:25,600 following the third booster. Purified IgG (1.8 mg/mL) exhibited high sensitivity in ELISA, detecting the recombinant β-SNTX at concentrations as low as 31.25 ng. The recombinant β-SNTX subunit demonstrated strong immunogenicity, inducing high-affinity antibodies with specific binding activity against the native toxin. The resulting antiserum demonstrated significant neutralization potential, highlighting its promise for antivenom development. |
| title | Bioinformatic Analysis and Recombinant Expression of the Stonustoxin β-Subunit for Polyclonal Antibody Development. |
| topic | Animals Rabbits Recombinant Proteins Computational Biology Escherichia coli Antibodies, Neutralizing Epitope Mapping Protein Subunits Gene Expression |
| url | https://pubmed.ncbi.nlm.nih.gov/41025585/ |