Wang Shuangshuang, Yang Yuxin, Yue Xinru, Liu Zewen, Yuan Fangyan, Yang Keli, Zhu Jiajia, Liu Wei, Tian Yongxiang, Wu Qiong, Gao Ting, Li Chang, Song Haofei, Zhou Danna, Bei Weicheng
National Key Laboratory of Agricultural Microbial Resources Discovery and Utilization, Huazhong Agricultural University, Wuhan 430070, China.
The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China.
Vet Sci. 2025 Apr 18;12(4):381. doi: 10.3390/vetsci12040381.
Enterotoxigenic (ETEC) is one of the primary pathogens causing diarrhea in piglets, causing significant economic losses in the swine farming industry. Due to the numerous serotypes of ETEC, traditional vaccines fail to provide sufficient cross-protection, and subunit vaccines based on epitope design have emerged as a safer and more effective approach for prevention and control. Unlike vaccine development strategies that involve the tandem arrangement of multiple antigenic epitopes, this study used the K88-FaeG protein as a backbone and incorporated the antigenic epitopes of K99-FanC to achieve a better immunogenicity. By using bioinformatics software to predict B-cell linear epitopes (score of over 0.6), B-cell epitopes from three-dimensional structures (50% amino acid score of ≥0.2), and B-cell epitope IgG antibody subtypes, as well as docking analysis with Sus scrofa aminopeptidase N (APN) receptors, six antigenic epitopes of K99-FanC were selected. Through Western blotting and competitive ELISA, we confirmed that all six recombinant proteins exhibited binding capabilities to K88- and K99-positive serum. The ELISA results showed that the serum levels of specific IgG and IgA antibodies increased after immunization, with FaeG-Ep3 and FaeG-Ep5 inducing the highest antibody titers against FanC-IgG (Log2 = 14.96) and FaeG-IgG (Log2 = 17.96), respectively. Bacterial adhesion assays revealed that only FaeG-Ep3 effectively blocked the adhesion of both K99 and K88 to IPEC-J2 cells. Immunization challenge experiments showed that, in the unimmunized group, mice infected with K88 and K99 experienced weight loss ( < 0.05) with intestinal villus shedding and intestinal wall structural damage. However, in the FaeG-Ep3-immunized group, no significant weight loss occurred after infection, and the villus protection rate (83%) was the same as that in the FaeG and FanC immunized groups. Overall, the FaeG-Ep3 recombinant protein identified in this study shows potential vaccine application value and provides new insights for developing multivalent vaccines against ETEC.
产肠毒素大肠杆菌（ETEC）是引起仔猪腹泻的主要病原体之一，给养猪业造成重大经济损失。由于ETEC血清型众多，传统疫苗无法提供足够的交叉保护，基于表位设计的亚单位疫苗已成为一种更安全、有效的预防和控制方法。与涉及多个抗原表位串联排列的疫苗开发策略不同，本研究以K88-FaeG蛋白为骨架，并入K99-FanC的抗原表位以获得更好的免疫原性。通过使用生物信息学软件预测B细胞线性表位（得分超过0.6）、三维结构中的B细胞表位（50%氨基酸得分≥0.2）以及B细胞表位IgG抗体亚型，以及与猪氨肽酶N（APN）受体的对接分析，选择了K99-FanC的六个抗原表位。通过蛋白质印迹法和竞争ELISA，我们证实所有六种重组蛋白均表现出与K88和K99阳性血清的结合能力。ELISA结果显示，免疫后特异性IgG和IgA抗体的血清水平升高，FaeG-Ep3和FaeG-Ep5分别诱导出针对FanC-IgG（Log2 = 14.96）和FaeG-IgG（Log2 = 17.96）的最高抗体滴度。细菌粘附试验表明，只有FaeG-Ep3有效阻断K99和K88对IPEC-J2细胞的粘附。免疫攻毒实验表明，在未免疫组中，感染K88和K99的小鼠体重减轻（<0.05），肠绒毛脱落，肠壁结构受损。然而，在FaeG-Ep3免疫组中，感染后未出现明显体重减轻，绒毛保护率（83%）与FaeG和FanC免疫组相同。总体而言，本研究中鉴定的FaeG-Ep3重组蛋白显示出潜在的疫苗应用价值，并为开发抗ETEC多价疫苗提供了新的思路。
