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由细菌引起的感染性腹泻,至今仍是世界范围广泛流行的传染病之一。由于耐抗生素病原菌的不断涌现,导致了抗生素药物的治疗效果不佳。因此,研发便捷、有效的疫苗对于细菌性腹泻的预防与治疗尤为重要。针对产肠毒素大肠杆菌、霍乱弧菌与志贺氏痢疾菌这三种最为主要的细菌性腹泻病原菌,设计和筛选了以热不稳定肠毒素亚基蛋白为抗原和黏膜佐剂、霍乱弧菌鞭毛蛋白及志贺毒素亚基蛋白为抗原的三联疫苗。通过工程大肠杆菌获得了相应的抗原与佐剂蛋白,并以海藻酸钙-壳聚糖微球为疫苗的口服载体,制备了疫苗的口服制剂。体外实验表明,微球载体中的蛋白质在模拟胃液中释放较低,但在模拟肠液中释放迅速,这种载体能够实现疫苗在肠道内定向释放的目的。通过对灌胃免疫后小鼠免疫指标的检测,证明了疫苗能够刺激机体产生抗原特异性的sIgA与IgG抗体,免疫组与对照组相比,差异显著(P<0.05),并提升了外周血中CD4~+T细胞的含量(7.5%~9.5%)与CD4~+T/CD8~+T细胞的比率,有效地激活了机体的黏膜免疫与系统免疫,能够对机体起到保护作用。
Infectious diarrhea caused by bacteria is still one of the most widespread infectious diseases in the world. Due to the continuous emergence of antibiotic-resistant pathogens, leading to poor treatment of antibiotic drugs. Therefore, the development of convenient and effective vaccines for the prevention and treatment of bacterial diarrhea is particularly important. In view of the three main pathogenic bacteria of bacterial diarrhea such as Enterotoxigenic Escherichia coli, Vibrio cholerae and Shigella dysenteriae, the heat-labile enterotoxin subunit protein as antigen and mucosal adjuvant was designed and screened. Vibrio cholerae Flagellin and Shiga toxin subunit proteins as antigen triple vaccine. The corresponding antigen and adjuvant protein were obtained through engineering E. coli, and the oral preparation of the vaccine was prepared by using calcium alginate-chitosan microspheres as oral carrier. In vitro experiments showed that the microspheres carrier protein release in the simulated gastric juice is low, but in the rapid release of simulated intestinal fluid, this vector can achieve the goal of targeted release of the vaccine in the intestine. The immunological indexes of mice after gavage immunization showed that the vaccine can stimulate the body to produce antigen-specific sIgA and IgG antibodies, the immune group compared with the control group, the difference was significant (P <0.05), and enhance the peripheral blood The ratio of CD4 ~ + T / CD8 ~ + T cells in CD4 ~ + T cells (7.5% ~ 9.5%) effectively activated the mucosal immunity and systemic immunity of the body and played a protective role on the body.