Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its Gene
Annual rept. 1 Mar 1986-28 Feb 1987
WYOMING UNIV LARAMIE DEPT OF MOLECULARBIOLOGY
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The purpose of this project is to 1 gain greater insight into crotoxin and crotoxin homolog structure, in order to provide a better understanding of this class of rattle-snake neurotoxin 2 develop an in vitro system for examining presynaptic neurotoxin mechanism of action 3 clone the crotoxin gene as a first step in creating a nontoxic, but immunoreactive crotoxin analog and 4 explore other possible non-toxic, crotoxin immunogens as potential vaccines against crotoxin and its homologs. We have raised polyclonal antibodies to crotoxin and its subunits from Crotalus durissus terrificus, Mojave toxin from Crotalus scutulatus scutulatus, concolor toxin from Crotalus viridis concolor, and monoclonal antibodies to crotoxin. We have used these to search for cross-reactive, homologous proteins in other venoms and examine their toxin neutralizing ability. The amino acid sequences of both crotoxin subunits were determined as a prelude to cloning. Based on this sequence information, we synthesized deoxyoligo-nucleotide probes complementary to the crotoxin gene to identify positive clones. Results with the polyclonal antibodies indicate that crotalid neurotoxins can be detected and neutralized by polyclonal antibodies raised against any intact toxin or basic subunit in this group of homologous toxins. Four different monoclonal antibodies have been purified and partially characterized, with one of these showing potent crotoxin neutralizing activity. This neutralizing monoclonal antibody is being used to raise anti-idiotype antibodies which will be examined for their potential as anti-idiotype vaccines.
- Genetic Engineering and Molecular Biology