众所周知,C.d.Terricus毒液会引起病理生理作用,例如神经病,凝血功能障碍,甚至死亡。先前的研究报道,ASC16可以与来自各种蛇类毒液的单体磷脂酶A2相互作用(例如,蛇形蛇和赤丝蛇)。因此,ASC16已被提议作为异二聚复合物(crotoxin)和C.d的毒液其他成分诱导的毒性作用的抑制剂。为了进一步调查,计算机模拟研究是使用crotoxin(CTX)蛋白复合物作为模型设计的,并进行实验测定以评估ASC16对CTX的抑制作用,以及其他毒液酶,如凝血酶样酶(TLE),磷酸二酯酶(PDE)和1-氨基氧化酶(LAAO)。对于体外测定,使用了特定的底物,使用体内小鼠实验模型(CF01)在48小时内测量致死活性。计算机研究表明,ASC16的亲水部分采用稳定的构象,同时与crotoxin的催化位点相互作用。在最高浓度下,ASC16显著抑制PLA2活性(40.89±0.09%),TLE(11.03±0.69%),PDE(51.33±2.83%),和LAAO(56.79±2.91%)。此外,ASC16中和了蛇毒的2LD50致死率。这些发现为设计有前途的佐剂奠定了基础,这些佐剂可以促进免疫方案中大量蛋白质的掺入。因此,这种方法旨在获得更高的抗体滴度,减少所需的免疫接种次数,并将生产动物的局部损害降至最低。
It is well known that C. d. terrificus venom causes pathophysiological effects such as neuropathies, coagulopathies, and even death. Previous studies have reported that ASC16 can interact with monomeric phospholipases A2 from the venom of various snake species (e.g., Vipera russelli and Echis carinatus). As a result, ASC16 has been proposed as an inhibitor of the toxic effects induced by the heterodimeric complex (
crotoxin) and other components of the venom of C. d. terrificus. To investigate this further, in silico studies were designed using the
crotoxin (CTX) protein complex as a model, and experimental assays were conducted to evaluate the inhibitory effect of ASC16 on CTX, as well as on other venom enzymes such as thrombin-like enzyme (TLE), phosphodiesterase (PDE) and l-aminoxidase (LAAO). For in vitro assays, specific substrates were used, and lethal activity was measured over 48 h using an in vivo murine experimental model (CF01). In silico studies have indicated that the hydrophilic portion of ASC16 adopts a stable conformation while interacting with the catalytic site of
crotoxin. At the highest concentrations, ASC16 significantly inhibited the activities of PLA2 (40.89 ± 0.09 %), TLE (11.03 ± 0.69 %), PDE (51.33 ± 2.83 %), and LAAO (56.79 ± 2.91 %). Furthermore, ASC16 neutralized the 2 LD50 lethality of crotalic venom. These findings lay the groundwork for designing promising adjuvants that can facilitate the incorporation of a larger quantity of proteins in immunization schemes. Consequently, this approach aims to achieve higher antibody titers, reduce the number of required immunizations, and minimize local damage in the producer animal.