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[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A]
Abstract:
The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.
Insights
Researchers developed a single-chain variable fragment (ScFv) antibody targeting Clostridium perfringens alpha-toxin. This recombinant ScFv protein effectively neutralized the toxin's phospholipase C activity in vitro.
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- Clostridium perfringens type A produces alpha-toxin, a potent phospholipase C, responsible for significant tissue damage.
- Development of targeted neutralizing agents against alpha-toxin is crucial for treating C. perfringens infections.
Purpose of the Study:
- To construct and express a recombinant single-chain variable fragment (ScFv) antibody targeting the alpha-toxin of Clostridium perfringens type A.
- To evaluate the neutralizing capability of the expressed ScFv protein against the toxin's enzymatic activity.
Main Methods:
- Amplification of VH and VL genes from a hybridoma cell line using RT-PCR.
- Construction of the ScFv gene by linking VH and VL domains with a flexible peptide linker.
- Cloning and high-level expression of the ScFv gene in E. coli XL1-BLUE.
- Analysis of ScFv expression using SDS-PAGE and functional neutralization assay.
Main Results:
- The ScFv gene, encoding 242 amino acid residues, was successfully constructed and cloned.
- High-level expression of the recombinant ScFv protein (approximately 25% of total bacterial protein) was achieved in E. coli.
- The expressed ScFv protein demonstrated significant neutralization of the phospholipase C activity of Clostridium perfringens alpha-toxin.
Conclusions:
- A functional recombinant ScFv antibody against Clostridium perfringens alpha-toxin has been successfully developed and expressed in E. coli.
- The expressed ScFv possesses potent neutralizing activity against the toxin's enzymatic function, indicating its therapeutic potential.
