Date of Award
Fall 12-11-2020
Document Type
Thesis
Degree Name
Master of Science (MS) in Biology
Department
Biology
Committee Chair
Dr. Christopher A. Murdock
Abstract
Snake Venom is a highly modified form of saliva, contains hundreds of zootoxins, necrotoxins, cytotoxins, neurotoxins, and mytotoxins, all of which are primarily made up of mostly proteins, polypeptides, and other components such as enzymes, lipids, and carbohydrates. Although snake venom is known for its harmful effects, it has also had a positive impact in scientific discovery and medicine. Some toxins from snakes function to inhibit pain, and this means they can be very effective analgesics. For instance, crotalphine is considered a 14-amino-acid-peptide that has a disulfide bond and shows analgesic properties through TRPA1 desensitization. This is the reason why snake venom peptides are at our center of interest; there are a plethora of new compounds to discover. Venomous snakes represent a large pool of potentially bioactive proteins, and despite recent computational studies and discovery efforts of over 2200 sequences, most of the snake venom toxins’ functions are still uncharacterized.
Using RNA pooled from the venom gland tissues of our sample, we constructed a cDNA library. In order to generate full-length transcripts, we used the SMARTTM technique for first-strand cDNA synthesis combined with duplex-specific nuclease enzyme from Kamchatka crab for cDNA normalization. Five hundred plaques were successfully screened, and 50 of them were sequenced. The plaques that we screened, amplified, and analyzed using agarose gel electrophoresis and then sequenced mostly coded for ribosomal proteins. Crotamine, C-type lectin (CLEC), phospholipase (PLA2), and serine protease were the components we were able to identify when we proceeded to annotate our 4 selected sequences with the venom-associated genes. Using predictive modeling, we determined specific locations, coding regions, variation information, exons, introns, and functions.
Crotamine functions to inhibit pain, and this means it can be a very effective analgesic; it is a cell-penetrating peptide and a suitable means for transporting macromolecules through cell membranes. Proteins with C-type lectin possess functions such as cell-to-cell adhesion, immune response to pathogens, and apoptosis and have also shown anticoagulant and procoagulant properties. PLA2 enzymes can bind to an array of proteins and target various types of tissues and organs, and they tend to cause myonecrosis and spastic hind-leg paralysis. Serine proteases play important roles in the diagnosis of hemorrhagic or thrombotic problems, and they stay in the human system for a clinically useful length of time.
The four identified components of the full-length-enriched cDNA of timber rattlesnake (Crotalus horridus) venom and their biochemical properties show promising applications in the field of pharmacology and medical sciences.