Snake Venomics Of Two Venomous Snakes And Application Of Species-specific Antibody

Snake venom is a complex mixture of toxic components, and produced in a specialized gland. Many venom components are bioactive proteins, which play important roles in capturing, killing and digesting prey. The variability of venom composition and content has attracted considerable scientific attention. With the development of techniques, studies of snake venomics have developed more quickly than ever before. Snakebites from snakes of the families Viperidae and Elapidae have long been a serious public health problem. However, many studies carried out to characterize the whole proteomes of snake venoms have focused on viperid snakes and, as such, less information has been available on the proteomes of venoms from elapid snakes.This study included two pieces of major work (here after Wl and W2):(W1) I studied the toxin profiles of Gloydius brevicaudus and Naja atra venoms by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, MALDI-TOF-TOF or CID-MS/MS. I also analyzed the enzymatic activities of both snake venoms.(W2) I studied the immunoreactivity between venoms and the commercial antiserums of G. brevicaudus, N. atra, Deinagkistrodon acutus, Bungarus multicinctus, and screened the species-specific antibodies to develop a ELISA test method for identification of venoms from these snakes. The following are the main results and conclusions of this study:W1Identification and enzymatic activities of snake venoms from G brevicaudus and N. atraSnake venoms from G. brevicaudus and N. atra could be fractionated clearly by RP-HPLC. The elution profile of G. brevicaudus venom proteins separated by RP-HPLC resolved27peaks. The elution profile of N. atra venom proteins separated by RP-HPLC resolved19peaks. Most of components could be identified by MALDI-TOF-TOF in both venoms, with a few components were identified by CID-MS/MS. The results showed that the snake venom of G. brevicaudus contains nine protein families:PⅡ-and PⅢ-SVMP (snake venom metalloproteases), D49-PLA2s (phospholipase A2), disintegrins, serine proteinases, LAO (L-amino acid oxidases), C-type lectins, CRISP (cysteine-rich secretory proteins) and5’-nucleotidases. The SVMP and PLA2represented64.5%and25%of total proteins in G. brevicaudus venom. Snake venom of N. atra contained four protein families:three-finger toxins, D49-PLA2s, CRISP and PⅢ-SVMP. The three-finger toxins and PLA2s represented83.1%and13.1%of total proteins in N. atra venom.The G. brevicaudus venom showed significant activity in hydrolyzing the casein, and about13-fold excess over that of N. atra venom and the5’-nucleotidases activity of N. atra venom was about27.5-fold of that induced by G. brevicaudus venom. The phosphomonoesterase activity was higher in N. atra venom, and was about3-fold higher than that in G. brevicaudus venom. The PLA2activity and LAO activity of G. brevicaudus venom presented a0.5-fold increase over that of N. atra venom. Moreover, the activity of serine proteases and fibrinolytic activity only occurred in G. brevicaudus venom. The acetylcholinesterase activity was only observed in N. atra venom.W2Preparation of species-specific antibodies and identification of snake venomsImmunoreactivity between four venoms and the corresponding commercial antiserums was evaluated by ELISA. The results revealed that positive signals could be detected between venoms and antiserums in all tests, and the cross-reactivity was increased when the concentration of antiserum was increased, but the reaction between homologous venom and antiserum was stronger than between heterologous venoms and antiserums. The assessment of immunoreactivity between venoms and antiserums was also carried out by western blot. The bands characteristics were corresponding to the protein molecules in SDS-PAGE. The component areas of venoms:low MW (Molecule Weights) bands (～14-35kDa) of Bungarus multicinctus, medium (～45-66kDa) and low MW bands (～14-25kDa) of N. atra, medium and high MW bands (～45-115kDa) of Da, and nearly all bands of G. brevicaudus showed high immunogenicity. Although the immunogenicity of most components in venoms was related to their abundance, there was no relationship observed between molecular sizes and immune response. In all cross-reactions between the venoms and antiserums, homologous venom and antiserum expressed highest reaction activity, the venoms and antiserums of species from same family presented higher activity than that from different family.The immunological specificity of species-specific antibody (SSAbs) from four types of commercial antiserums was determined by ELISA and western blot. The results showed in different concentrations, each of the four SSAbs was specific to one species. Snake venom identification was determined by ELISA. In all tests, the positive samples were identified successfully in about35min. The OD values of positive control and sample wells in identification of N. atra venom were about0.5and0.4, and were about1/2-2/3of that in the identification of other species. Four positive values of samples were much higher than the cutoff values, which were0.12,0.09,0.08and0.08.