Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM₁₉₇), a non-toxic mutant of diphtheria toxin. We derivatized CRM₁₉₇ and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM₁₉₇-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM₁₉₇ ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis.

志贺氏菌病是一种由肠道侵袭性细菌志贺氏菌引起的腹泻病，是儿童的主要问题，通常会导致死亡。由于这些菌株中的大多数已经产生了普遍的抗生素耐药性，因此开发疫苗对于对抗感染至关重要。来自S. flexneri 2a 型的 O 特异性多糖 (O-PSs)被认为是志贺氏菌病的主要致病抗原。因此，与载体蛋白结合的 O-PSs 可以作为潜在的高分子量疫苗候选者。因此，在本研究中，从S. flexneri 2a 中提取的 O-PS与交叉反应材料 (CRM ₁₉₇ ) 结合，这是一种白喉毒素的无毒突变体。我们衍生了 CRM ₁₉₇和 O-PS 分别与己二酸二酰肼 (ADH) 和它们的对应物反应以探测缀合功效。在这两种策略中，用 O-PS 处理的 CRM ₁₉₇ -ADH 产生了 311 kDa 的稳定糖缀合物。通过使用狭缝印迹、尺寸排阻和高性能阴离子交换色谱技术估计游离蛋白质、游离 O-PS 和 O-PS:CRM ₁₉₇比率来探测缀合效率。该缀合物表现出三个月的延长的保质期。Vero/MRC-5 细胞的细胞毒性研究证实了偶联物的无毒性，这使得该糖偶联物成为志贺氏菌病的潜在候选疫苗。