| Tetrodotoxin (TTX) is a small molecular weight guanidinium neurotoxin thatoccludes voltage-gated sodium channels in nerve and muscle tissue, resulting inrespiratory paralysis and death. The effective method for TTX detection is stilllacking, so it is very important to develop an immunological method for TTXdetection. In the present study, we used a chemical method (Mannich reaction) toprepare the tetrodotoxin complete antigen (TTX-BSA as the detection antigen andTTX-KLH as the immunity antigen), and used a small mount, repeatedly immunityway to immunize3Balb/c. The variable regions of heavy chain (V_H) and light chain(V_L) were amplified from the first-strand cDNA that was synthesized throughRT-PCR from the spleen mRNA through the primary PCR amplification. Arecombination scFv gene that was composed of V_H, V_L, and linker was formated by aSOE-PCR. A murine phage display library was constructed in a pCANTAB-5E phageplasmid vector. Phage particles displaying scFv were rescued by reinfection of helperphage M13kO7followed by six rounds of biopanning for selection of TTX bindingscFv antibody fragments by using phage enzyme-linked immunosorbent assay(ELISA). Soluble TTX-scFv-T53antibodies were characterized from one of theclones showing the highest affinity to TTX, and its affinity constant was1.1x106L/mol. The gene sequence and amino acid sequence of the selected clone wereanalyzed, and the division map of CDRs, secondary structure and physical andchemical properties were predicted by the means of bioinformatics. These resultsdemonstrated that the recombinant antibody constructed by immortalizing theantibody genes was successful. |
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