关键词: 克伦特罗, 单链抗体, 结构预测

Abstract:

Objective: To construct the gene of single chain Fv (scFv) fragment against clenbuterol, to predict secondary and tertiary structures of its encoding protein by computer assisted modeling and to analyze its physical and chemical properties. Methods: The gene of clenbuterol scFv was constructed with 5D1 hybridoma cells that is able to secret monoclonal antibodies (mAb) with high activity and specificity against clenbuterol as the raw material, and sequenced by splicing overlap extension (SOE) PCR assay. Furthermore, its amino acid sequence was deduced and the secondary and tertiary structures were predicted using biological information software. Meanwhile, its physical and chemical properties were analyzed. Results: The full-length scFv gene had 720 bp and encoded 225 amino acids. The relative molecular weight of the scFv was 25826.6, its predictive isoelectric point (pI) was 8.78 and the antibody belonged to alkaline protein. The predicted secondary structure showed that the protein contained 20 α-helix, 99 β-sheet, 28 β-turn and 93 random coli. The mimic tertiary structure model of the scFv indicted that the VL and VH was involved in composing the hydrophobic“ pocket” which was beneficial to the antigen binding, but the linker was free from this structure. Conclusion: The successful construction of a scFv gene cbl against clenbuterol by us can laid the foundation for the further research into the expression, purification and bioactivity of genetic engineering antibody.

Key words: clenbuterol, single chain Fv (scFv), structure prediction