Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

中文翻译：

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含有crotoxin表位的工程蛋白可在免疫兔中诱导中和抗体。

Crotoxin（Ctx）是响尾蛇毒的主要致死成分。它是一种神经毒素，由两个通过非共价相互作用相关联的亚基组成，分别是无毒的酸亚基（Crotapotin）和基本亚基（CB），具有磷脂酶A2（PLA2）活性。利用SPOT合成技术，我们确定了位于每个Ctx亚基C末端的两个表位。另外，使用通过手术插入小鼠的皮下斑点植入物，将3个其他表位定位在Ctx的不同区域。此处定位的所有表位均以重组多表位蛋白（rMEPCtx）形式表达，用于免疫新西兰兔。抗rMEPCtx兔血清与Ctx和C.d.的粗毒液交叉反应。特异种，Crotarus durissus ruruima，秘鲁C.durissus和Bothrops jararaca（强度较低）。此外，抗rMEPCtx血清能够中和Ctx致死活性。由于重组多表位蛋白无毒，因此可以大剂量给药而不会对免疫动物的健康造成不利影响。因此，我们的工作证明了Ctx中和表位的鉴定，并支持使用重组多表位蛋白作为免疫疗法生产的创新。