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【目的】比较小鼠滴鼻与口服接种ETEC F41重组干酪乳杆菌后,机体所产生的抗ETEC F41的黏膜免疫和系统免疫及免疫保护率的差异,为确定ETEC乳酸菌疫苗免疫程序奠定基础。【方法】将构建的重组质粒pLA-F41电转化入干酪乳杆菌,获得阳性重组菌。重组菌在MRS培养基中进行表达,经Western blot检测目的蛋白的表达,间接免疫荧光及流式细胞术检测外源蛋白展示到菌体表面。SPF级BALB/c小鼠随机分成4组,每组40只,将重组菌以滴鼻和口服途径分别接种2组小鼠,对照组分别接种同剂量的空质粒菌株,免疫4次,初免后不同时间采集血液样品,测定小鼠产生抗F41的特异性IgG,收集小鼠肺部冲洗液、肠道冲洗液、阴道冲洗液及粪便样品测定小鼠产生抗F41的特异性sIgA,并对主动免疫和被动免疫小鼠进行攻毒保护性试验。【结果】重组干酪乳杆菌pLA-F41/L.casei免疫小鼠能够产生明显的抗F41的IgG和sIgA抗体水平,口服主动免疫组保护率在90%以上,滴鼻主动免疫组保护率在85%以上,对照组均全部死亡。口服被动免疫组保护率达90%,滴鼻被动免疫组保护率达80%,对照组保护率均为5%。【结论】滴鼻免疫和口服免疫重组干酪乳杆菌后能产生相近的抗ETEC F41的黏膜免疫和系统免疫并能有效预防ETECF41的感染。
【Objective】 To compare the mucosal immunity, systemic immunity and immune protection rate of ETEC F41 induced by ETEC F41 intranasally inoculated intranasally and orally in mice, which lays the foundation for the identification of ETEC lactic acid bacteria vaccine. 【Method】 The recombinant plasmid pLA-F41 was transformed into Lactobacillus casei to obtain a positive recombinant strain. Recombinant bacteria were expressed in MRS medium. The expression of the target protein was detected by Western blot. The foreign proteins were detected by indirect immunofluorescence and flow cytometry. SPF grade BALB / c mice were randomly divided into 4 groups, 40 in each group. The recombinant strains were inoculated intranasally and orally respectively to 2 groups of mice. The control group were inoculated with the same dose of empty plasmid strains respectively and immunized 4 times. Blood samples were collected at different times, the mice were tested for anti-F41-specific IgG, mouse lung washings, intestinal washings, vaginal washings and stool samples were collected to determine the specific anti-F41-specific sIgA in mice Active immunization and passive immunization of mice for challenge protection test. 【Results】 The recombinant Lactobacillus casei pLA-F41 / L.casei immunized mice can produce significant anti-F41 IgG and sIgA antibody levels, oral active immunization group protection rate of 90% or more, nasal active immunization group protection rate of 85 %, The control group all died. Oral passive immunization group protection rate of 90%, nasal passive immunization group protection rate of 80%, the control group were 5% protection rate. 【Conclusion】 Intranasal immunization and oral immunization of recombinant Lactobacillus casei can produce similar mucosal immunity and systemic immunity to ETEC F41 and can effectively prevent ETECF41 infection.