King Cobra Three Refers To The Toxin Gene Cloning, Expression And Function Of Recombinant Long-chain Neurotoxin

Three-finger toxins are a family of low molecular weight toxins (<10kDa) having a very similar tertiary structure. We designed the degenerative primers based on the highly conversed residue sequences of α-neurotoxins and obtained partial DNA sequences of neurotoxins from Ophiophagus Hannah. Followed by 3'RACE and RT-PCR, 19 novel cDNAs of three-finger toxins containing the sequence of signal peptide and 3'-UTR region were cloned from the venom glands of O.Hannah. Putative peptides encoded by the cDNAs were classified as six kinds of three-finger toxins: long chain neurotoxins, short chain neurotoxins, weak neurotoxins, cardiotoxins, muscarinic toxins and a toxin with a free SH group. It indicated that the accelated envolution and positive Darwinian selection had taken place in those genes according to the mathematical analysis based on the synonymous and non-synonymous substitutions in the coding and non-coding regions. Phylogenetic trees were constructed to describe the evolutionary relationship of these toxins in a specific snake. It also suggested that long chain neurotoxins of O.Hannah had closer evolutionary relations with short chain neurotoxins of HYDROPHIIDAE.According to the sequence alignment, we chose 14 cDNAs from the 19 genes and expressed them in E.coli. It shows that all the toxins could be expressed in a high level and most of them had the soluable forms except the cardiotoxins. We optimized the conditions of expression and digestion of two long chain neurotoxins and established the process of purifying recombinant toxins from the cleavage buffer by the GST column and the Benzamidine column connected in series. Recombinant peptides were characterized by MALDI-TOF mass, N-terminal residue sequencing, Tris/tricine SDS-PAGE and CD spectrometry. The purity of them was tested by RP-HPLC and SEC-HPLC.The results of LD₅₀ test and whole-cell patch clamp technology showed that recombinant toxins had potent neurotoxic acitivity and could be classified into postsynaptic neurotoxins. Whereas, some interesting phenomena observed in cloning and expressing stages and in LD₅₀ test indicated the existence of cardiotoxin-like cytolytic activity in these two recombinant peptides. Results of the MTT assay on HUV-EC and L929 cells confirmed this conjecture. By comparing the functional residues, we offered some possible explanations for the difference of their neurotoxic functions. Moreover, plausible elucidation on the extra cytolytic activity was carried out through the analysis of a hydropathy profile.