Construction And Exression Of Anti-curcumol ScFv In E.Coli

Objective: To produce high affinity anti-curcumol single-chainvariable fragment antibody, by using gene engineering methods.Methods: The genes encoding light chain and heavy chain ofanti-curcumol antibody were cloned by RT-PCR and regular molecularcloning methods. Overlapping extension PCR method was employed toconstruct anti-curcumol single chain variable fragment (ScFv) gene thatwas subsequently expressed in bacterial cells. The activity of expressedScFv protein is detected by ELISA assay.Result: Genes encoding antibody light chain and heavy chainvariable region were amplified from hybridoma cells and cloned intoT-easy vector. Sequencing results showed we get new genes encodingantibody light and heavy chain that have not been reported in Genebank.Gene encoding ScFv antibody was constructed by introducing a sequenceencoding polyglycine linker between light chain variable region andheavy chain variable region genes using overlapping extension PCR methods. The ScFv gene was subsequently cloned into bacterialexpression plasmid to get plasmid of pET21-scFvFH that was sequencedand confirmed to have the right expression cassette producinganti-curcumol scFv with a C-terminal flag tag and6XHis tag. Theplasmid of pET21-scFvFH was transduced into Rosseta cells and inducedto expression ScFv protein. The IPTG stimulated bacterial were harvestedfor protein extraction and Western blot assay using anti-flag antibody.The results showed that around95%of ScFv protein was in inclusionstate, while the other5%of ScFv protein was soluble protein that hadhigh activity to specifically bind with curcumol, as detected by ELISA.Conclusion: We successfully cloned genes encoding variable regiongenes of anti-curcumol antibody from hybridoma cells. Anti-curcumolscFv were constructed and expressed in bacterial cells and producedhigh activity scFv antibody specifically recognizing curcumol. This worksupporting the later curcumol quality control and screening of highcurcumol production strains to promote the application of natureanti-tumor compound.