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目的评估D蛋白(protein D,PD)黏膜免疫对流感嗜血杆菌感染的保护效果,研究其免疫应答机制,为发展新疫苗提供依据。方法运用分子克隆技术构建PD蛋白表达质粒;用含有或无霍乱毒素(cholera toxin,CT)的PD蛋白黏膜免疫C57BL/6小鼠,ELISA方法检测血清Ig G、唾液Ig A及脾细胞的细胞因子分泌水平;建立定植和致死性感染模型,观察细菌清除及小鼠生存情况;以裸鼠和μMT小鼠来研究细胞和体液免疫应答在PD免疫保护效果中的作用。结果单独PD蛋白黏膜免疫野生鼠能诱导高滴度的抗PD Ig G和s Ig A,血清中Ig G1、Ig G2a和Ig G2b亚型水平相当,脾细胞分泌IL-17A增加,小鼠鼻咽部流感嗜血杆菌清除增加,致死性感染生存率为82%;裸鼠和μMT小鼠免疫PD后对流感嗜血杆菌的清除效应和致死性感染保护作用受损,PD抗血清被动免疫μMT小鼠后其抗感染能力恢复;PD+CT组抗体滴度及脾细胞分泌IL-4和IL-17A水平均高于PD组,细菌清除效应与PD组相似,但无致死性感染保护能力。结论无佐剂PD蛋白黏膜免疫能有效诱导黏膜应答,抵抗流感嗜血杆菌的定植与致死性感染,为黏膜疫苗的发展提供了可能的选择。
Objective To evaluate the protective effect of mucosal immunity of protein D (PD) on Haemophilus influenzae infection and to study its mechanism of immune response to provide a basis for the development of new vaccines. Methods PD protein expression plasmids were constructed by molecular cloning technique. C57BL / 6 mice were immunized with PD protein mucosa with or without cholera toxin (CT), and serum Ig G, salivary Ig A and cytokines The model of colonization and lethal infection was established to observe the bacterial clearance and the survival of mice. The effects of cellular and humoral immune responses on the protective effect of PD were studied in nude mice and μMT mice. RESULTS: Immunization with wild-type PD mouse mucosal immunized mice induced high titer of anti-PD Ig G and s Ig A. The serum Ig Ig, Ig G2a and Ig G2b subtypes were comparable, the splenocytes secreted IL-17A increased, Department of Haemophilus influenzae increased clearance, fatal infection survival rate was 82%; nude mice and μMT mice after immunization of PD Haemophilus influenzae clearance and lethal infection, protective effect of impaired, PD antisera passive immunization μMT small The antibody titer in PD + CT group and the levels of IL-4 and IL-17A secreted by spleen cells in PD + CT group were higher than those in PD group. The bacterial clearance effect was similar to that in PD group, but no protection against lethal infection. Conclusion Mucosal immunization without adjuvant PD protein can effectively induce mucosal response and resist the colonization and lethal infection of Haemophilus influenzae, which may provide a possible alternative for the development of mucosal vaccine.