Purification and characterization of insect toxins from the venom of the scorpion Buthus occitanus tunetanus
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文摘
The venoms of Buthidae scorpions are known to contain single basic chain proteins of 60-70 amino acid residues tightly reticulated by four disulfide bridges. The acute toxicity of these proteins is due to their high binding affinity to voltage-sensitive sodium channels, thus impairing the initial rapid depolarization phase of the action potential, in nerve and muscle. Many studies have focused on the purification and characterization of the structural and immunochemical relationships of Buthus occitanus tunetanus (Bot) scorpion neurotoxins. Neurotoxins purified from Bot venom are α-type toxins. For improved serotherapy of Buthidae scorpion stings as well as the development of immunotherapy approach, we started the construction of a cDNA library of Bot scorpion using recombinant DNA techniques, in order to clone and express toxin genes and to explore structure-activity relationships. As a first step, we analysed clones from cDNA library by two oligonucleotides probes with sequence homology to regions coding for toxins. Among the six recombinant clones, we selected the present nucleotide sequence of the cDNA 4E4, an unexpected amino acid sequence. Indeed, it contains a high number of histidine residues. In consequence, we decided to express it and to determine its toxicity and antigenicity. Thus, we have shown that scorpion neurotoxin can be successfully synthesized as a fusion protein. In this communication, we firstly describe the construction of the recombinant expression plasmid encoding a proteinA-protein4E4 fusion protein. The sequence of the mature protein has no methionine residue, thus, we introduced a site potentially cleavable by cyanogen bromide at position −1 of the protein sequence. Thus, the recombinant protein would be regenerated by chemical cleavage. Secondly, we show that the hybrid is directly secreted in the periplasm ofE. coli in the milligram range per litre of culture (> 10 mg/litre of culture). Thirdly, the hybrid protein and the cleaved recombinant protein is recognized by anti-Bot G50 and anti-BotI antibodies and not recognized by anti-AaHII antibodies. Studies are underway for further biochemical and immunological characterization. An immunization programme is also being undertaken to evaluate in mice the protection and neutralization effects of sera produced by the recombinant toxin. This recombinant hybrid could be used for antiscorpionic vaccinotherapy.