Cloning, sequence analysis and expression of a single-chain antibody against clenbuterol
July 12 18:00:46, 2025
Total RNA was extracted from a hybridoma cell line that secretes anti-Clenbuterol monoclonal antibodies. The variable heavy (vH) and variable light (vL) chains were amplified using reverse transcription-polymerase chain reaction (RT-PCR). The vH and vL fragments were then ligated into a single-chain variable fragment (ScFv) using a flexible peptide linker (GGGGS). Following sequencing, the resulting ScFv was analyzed using NCBI Blast to confirm its identity as a recombinant functional murine antibody variable region gene. The target gene was subsequently cloned into the expression vector pET-22b(+) and transformed into *E. coli* BL21 competent cells. Protein expression was induced with IPTG under different temperature conditions: 37°C for 5 hours and 25°C for optimal solubility. The expressed protein was analyzed by SDS-PAGE to evaluate its size and purity. The results showed that at 25°C, the protein was predominantly soluble, while at 37°C, the expression level was higher but less soluble. This study provides a foundation for further optimization of the ScFv expression system for potential applications in immunoassays or therapeutic development.
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