Construction Of Anti-tetrodoxotin Single Chain Antibody Varible Fragment (scFv)

Tetrodotoxin (tetrodotoxin, TTX), a small molecule, non-protein, highly toxic toxin, can cause human beings and animals dead if it is eaten by mistake. TTX is a typical sodium ion channel inhibitor, can led to limb numbness, paralysis and other symptoms, even seriously to death. Human lethal dose of TTX is about 0.5mg/70kg, but we lack simple and effective detection methods for TTX so far. In this study, we obtained genetically engineered single-chain antibody against TTX for the preparation of TTX detection kit.TTX, whose molecular weight is only 319.26, has only reactivity and can not provoke immune system. So TTX was connected to keyhole limpet hemocyanin (KLH) by chemical method to construct complete antigen, which had immunogenicity and then was used as antigen. TTX was also connected to bovine serum albumin (BSA) for detection. Healthy Balb/c female mice of 6-8 weeks age were used for immunization. After several times strengthening immune, mice serum titer was tested. Once the serum titer reached a high value, mice was sacrificed and total RNA was extracted from the spleens.The first cDNA strand was synthesized by RT-PCR from mRNA. Then VH (heavy chain variable region) and VL (light chain variable region) were obtained from cDNA template by PCR. Subsequently, a nucleotide sequence which encode the small peptide (Gly4Ser)3 was used as a linker to construct scFv gene by overlap extension PCR. Then scFv gene was cloned into the phage vector pCANTAB-5E to construct a single-chain phage antibody library. After a panning of six rounds from the library, a phage clone WY5 with high affinity was obtained.