The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.

中文翻译：

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使用枯草芽孢杆菌表达的重组rCpa-bx对小鼠产气荚膜梭状芽孢杆菌的α，β和ε毒素进行免疫。

厌氧性产气荚膜梭状芽胞杆菌是肠道疾病的最有力原因，例如肠道毒素血症，出血性肠炎和羊痢疾。三种毒素型（B，C和D）通常是造成这些疾病的原因，并且主要通过三种重要的外毒素介导：α毒素（CPA），β毒素（CPB）和epsilon毒素（ETX）。根据结构疫苗学的原理，我们设计了一种嵌合蛋白rCpa-bx，它包含CPA的C末端结合区，CPB的部分序列和ETX（分别为Cpa247-370，Cpb108-305和EtxH118P） 。然后，使用枯草芽孢杆菌作为递送载体（Bs-pHT43-Cpa-bx）通过pHT43质粒在体内表达rCpa-bx蛋白。rCpa-bx蛋白的免疫活性通过Western印迹验证，并在鼠模型中评估其免疫效力。口服重组试剂引起局部粘膜和全身免疫反应，血清IgG和肠粘膜分泌性IgA（sIgA）抗体滴度显着增加。与对照组相比，从Bs-pHT43-Cpa-bx组分离的淋巴细胞中IL-2，IL-4和IFN-γ的水平明显更高。Bs-pHT43-Cpa-bx组和灭活疫苗（IV）组中CD4 +和CD8 + T淋巴细胞的百分比在正常范围内。疫苗组和对照组的小鼠用含α，β和ε毒素的1x LD100单位滤液攻击。发现Bs-pHT43-Cpa-bx组的小鼠发病率较低。在10天观察期结束时，用Bs-pHT43-Cpa-bx进行的主动免疫小鼠仍保持85％至90％的存活率，而对照组小鼠则在2至5天内死亡。这项研究的结果证明Bs-pHT43-Cpa-bx可以预防小鼠产气荚膜梭菌感染，并且Bs-pHT43-Cpa-bx可以被认为是针对产气荚膜梭菌的潜在疫苗。