Najaatra,中国眼镜蛇,是亚洲蛇毒的主要原因,每年造成成千上万的临床事件。目前的治疗,马血清来源的抗蛇毒血清,具有不可预测的副作用,并提出了制造挑战。这项研究的重点是通过使用微生物噬菌体展示技术从用减毒N.atra毒免疫的羊驼中获得纳米抗体来开发新一代蛇毒解毒剂。在确认羊驼的免疫反应后,我们从分离的外周血单核细胞中扩增了VHH基因,并构建了1.0×107个转化体的噬菌体展示VHH文库。经过四轮生物淘选,富集的噬菌体对N.atra毒液的结合活性增加。选择具有高结合亲和力的四个纳米抗体克隆:aNAH1、aNAH6、aNAH7和aNAH9。对各种蛇类毒液的特异性测试,包括两种东南亚的眼镜蛇,揭示了眼镜蛇属特有的纳米抗体。体内小鼠毒液中和试验表明,所有纳米抗体都延长了小鼠的存活时间,aNAH6保护了66.6%的小鼠免于致死剂量。这些发现强调了噬菌体展示衍生的纳米抗体作为有价值的解毒剂的潜力。为未来在蛇咬伤治疗中的应用奠定基础。重要中国眼镜蛇的毒液咬伤带来了巨大的医学挑战,和目前的血清治疗面临尚未解决的问题。我们的研究应用微生物噬菌体展示技术获得了一种新的,有效,和具有成本效益的治疗方法。尽管科学家对利用这项技术筛选羊驼抗毒素抗体感兴趣,现有文献有限。这项研究通过引入专门针对中国眼镜蛇毒的中和抗体做出了重大贡献。我们对抗体构建过程提供了全面而公正的说明,伴随着各种纳米抗体的全面测试和与不同蛇毒的交叉反应性的评估。这些纳米抗体代表了有针对性的抗蛇毒血清开发的有希望的途径,它将微生物学和生物技术连接起来,以满足关键的健康需求。
Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive
nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed
nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all
nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived
nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These
nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.