产气荚膜梭菌 (C. perfringens) 的所有亚型都会产生 α 毒素 (CPA)，可引起羔羊、牛、猪和马的肠炎或肠毒血症，以及人类和动物的创伤性梭菌性肌坏死。 CPA 作用于细胞膜，最终导致内吞作用和细胞死亡。因此，中和CPA对于预防和治疗产气荚膜梭菌引起的疾病至关重要。本研究以CPA为抗原，从天然产物中制备了半衰期为2.9 h、亲和常数（KD）为0.9 nmol/L、在60℃以下稳定性良好的纳米抗体（CPA-VHH）。来自羊驼的纳米抗体库。 CPA-VHH的生物活性分析表明其能够以15倍的比例有效中和磷脂酶和CPA的溶血活性。在 Vero 细胞中，9.8 μg/mL CPA-VHH 在两倍半数抑制浓度 (IC50) 下中和了 CPA 的细胞毒性。在小鼠模型中，35.7 ng/g 体重 (BW) 的 CPA-VHH 中和了 2 倍半数致死剂量 (LD50) 的 CPA 引起的 90% 的致死率。结果发现，在 2 × LD50 CPA 下，CPA-VHH 在 30 分钟内保护了 80% 的小鼠，但在 2 小时后下降到 50% 以下，在 4 小时后降至 0%。抢救试验表明，感染后30分钟内使用2×LD50 CPA的CPA-VHH可实现80%的抢救率，2小时后降至10%。此外，CPA-VHH 有效减轻了 zonula occlusionns-1 (ZO-1)、Occludin 和 Claudin-1 表达水平的降低，同时还减弱了促炎细胞因子白介素-1β (IL-1β) 的上调CPA 诱导的白细胞介素 6 (IL-6)、白细胞介素 7 (IL-7)、白细胞介素 8 (IL-8)、肿瘤坏死因子 α (TNF-α) 和干扰素 γ (IFN-γ)感染。总体而言，本研究确定了一种特定的纳米抗体CPA-VHH，它可以在体外和动物模型中有效中和CPA毒素，为抑制这些毒素产生的致病性提供了新工具，并为开发新型抗毒素奠定了重要基础。 C.产气荚膜毒素相关的治疗产品。

All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 μg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC50). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD50) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD50 CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD50 CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products.