Selecting Mimotope Of Tetrodotoxin By Phage Display Techniques

Tetrodotoxin (TTX) is a kind of non-protein marine bioactive substance which is strongly poisonous. TTX acts on the central and peripheral nervous system, binding selectively with the protein of sodium ion channel on cell membrane, thereby blocking sodium channel, then directly affecting the excitability of neuromuscular transmission, making neuromuscular paralysis and even lead to death. It has stable physical and chemical properties so that high temperature, solarization and normal cooking process cannot break it down. Tetrodotoxin can be used in clinical analgesic, anesthetic and blood pressure medicine, etc., but its content is hard to grasp. The most commonly used detection methods of tetrodotoxin are physical and chemical analysis and immunochemical methods, but both of them have limitations, one of which is that they must use TTX standard. Currently, TTX standard is very expensive and international embargo as a biological warfare agent. In addition, TTX is a highly toxic material to the operators. Therefore, if we find a substance so if it can substitute for tetrodotoxin and identify its nature, whether for drug-free detection method, or as a prerequisite for drug screening, all have a greater significance.Some scholars use the anti-TTX monoclonal antibody anti-idiotypic antibodies (AID) substitute for tetrodotoxin in ELISA, but the affinity between anti-idiotypic antibody and antibodies is usually higher than the affinity between antibody and toxin, so the AID cannot substitute bound TTX which coated microtiter plate for ELISA, and the preparation of anti-idiotypic antibodies is very cumbersome. Phage random peptide library technology is an effective solution to this problem. Phage random peptide library is a large number of random encoded peptide sequences into phage vector to create phage display library, each phage particle surface only demonstrate a sequence of exogenous peptide, and maintain their relative conformation and biological activity. In this study, we use anti-TTX monoclonal antibody as the target molecule for biopanning from a phage random peptide library, and then synthesize polypeptide based on the TTX mimic epitope and to substitute TTX standard, which can set up a base for the establishment of non-toxic ELISA detection.The monoclonal antibody against the TTX was used as ligand to screen the binding peptide from the Ph.D.-7 peptide library. This library is displayed as a fusion protein with the coat protein III of filamentous phage M13. The positive clones were identified by ELISA. The peptide sequences of positive phage clones were determined and analyzed by DNA sequencing, then chemically synthesized peptide to do further validation. After four rounds of panning,7 positive clones can binding to the antibody, and through indirect competitive ELISA,3 positive clones could inhibited TTX. A competitive ELISA was established with clone P4, the linear of the inhibition was between lng/ml and 20ng/ml, R2=0.9947, the detecting limitation was 1ng/ml, and 50% inhibition rate was 4ng/ml. There was no difference between this method and the conventional ELISA detection method while detecting some samples. Result of DNA analysis showed that the common amino acid sequences were Histidine-Glycine-Proline-Tyrosine-Arginine-Histidine-Proline (HGPYRHP).According to DNA sequencing analysis result, synthetic peptides (HGPYRHP), and establish immunoassay, the linear range was 1-20μg/ml, R2=0.9995,20% inhibition rate was 1.2μg/ml,50% inhibition was 6.3μg/ml.