艰难梭菌是医院内感染的主要病原菌之一，近年来，随着高致病株的出现和菌株耐药性的增加，艰难梭菌的感染在全球呈流行趋势，由于毒素是其主要的感染因子，且病变部位是肠道，因此，研制粘膜免疫效果良好的类毒素疫苗是防治艰难梭菌感染的关键。本项目利用疫苗免疫原理和技术，首先通过艰难梭菌透析培养、盐析、离子交换层析等方法纯化艰难梭菌A、B毒素，经脱毒后分别制备艰难梭菌疫苗(类毒素A，类毒素B和类毒素A+B)，与粘膜佐剂(CT/LT)配伍后，按特定的免疫程序，采用微针技术经皮免疫BALB小鼠，定期检测特异性血清IgG和粪便中S-IgA的效价水平变化，最后采用艰难梭菌动物感染模型和细胞检测来评价疫苗的实际免疫保护效果，通过分析不同佐剂对免疫效果的影响，确立艰难梭菌疫苗最有效的粘膜免疫佐剂、最佳类毒素抗原种类和配比，揭示肠道粘膜免疫水平在细菌感染中的作用，为艰难梭菌类毒素疫苗的研制提供技术和理论参考。

Clostridium difficile infection(CDI) has been identified as the leading cause of nosocomial infection. With the emergence of hypervirulent strains and the strains resistant to metronidazole or vancomycin in recent years, the prevalence and severity of CDI has increased in the world. As C. difficile toxins A and B play important roles in infection as well as in gut inflammation, the highly effective mucosal toxoid vaccines are necessary for CDI control. According to the immunological principle and technology of vaccine preparations, first of all, the C.difficile are cultured anaerobically by the dialysis bag methods, the toxin A and toxin B will be purified by precipitation with (NH4)2SO4 followed by ion-exchange chromatography. C. difficile toxoids vaccine are prepared with formaldehyde-inactivated the C. difficile toxins A,toxin B and toxin A+B). Secondly, the female BALB/C mice will be transcutaneous immunized with or without cholera toxin(CT) and E. coli heat-labile enterotoxin (LT) as mucosal adjuvant respectively by microneedle patches. And then, in order to detect the systemic and mcosal immune responses, the specific serum immunoglobulin (IgG) and secretory IgA (S-IgA) in stool against to the C. difficile toxins will be measured by enzyme-linked immunosorbent assay. Finally, the immunogenicity and protective activity of the vaccine will be examined in the mice C. difficile challenge model, Serum from individual animals was tested for neutralizing activity against native C. difficile toxin A and toxin B in the Vero cell based neutralization assay. By analysis of the immune effects of different adjuvants used, not only the most effective mucosal adjuvants, optimal antigen ratio of C. difficile toxoid vaccine could be established, and the role played by intestinal mucosal immunity in the occurrence and change of the CDI will be revealed, but also the study will offer technology and biological data for C.difficile toxoid vaccine candidate for human use.