减毒沙门菌为载体的PspA优势抗原组合DNA疫苗对肺炎链球菌定植的防御作用评价  

Protection effects of DNA vaccine with multiple pspA genes against streptococcus pneumonia nasopharyngeal colonization using the salmonella-based new carrier system

作　　者：张巧[1] 刘进[1] 刘聚伟[2] 万敏[1] 吴颖[1] 李瑾[1] 王长征[1] 马千里[1] 

机构地区：[1]第三军医大学附属新桥医院全军呼吸内科研究所,重庆400037 [2]解放军第309医院西山门诊部,北京100091

出　　处：《中华肺部疾病杂志（电子版）》2013年第5期34-39,共6页Chinese Journal of Lung Diseases(Electronic Edition)

基　　金：重庆市自然科学基金(2010BB5181)

摘　　要：目的评价以减毒沙门菌为载体携带PspA不同家族亚类优势抗原的肺炎链球菌DNA疫苗对不同侵袭性肺炎链球菌定植的防御作用,探讨优化肺炎链球菌DNA疫苗增强鼻咽部黏膜免疫的策略。方法应用分子克隆技术,在前期研究基础上,制备携带PspA不同家族亚类优势抗原(PspA-Fam1-Clade1和PspA-Fam2-Clade3)的减毒沙门菌为宿主菌的非抗性基因筛选的真核质粒-宿主致死平衡系统,应用RT-PCR和免疫印迹技术检测优势抗原的表达情况,并在含或不含DAP的SOB培养基中传代培养,抽提质粒PCR检测疫苗携带抗原的稳定性。以PBS为对照,口服免疫接种BABL/c小鼠,ELISA检测鼻咽部sIgA的水平;应用小鼠鼻咽部定植模型检测疫苗对不同侵袭性肺炎链球菌定植的防御作用。结果成功构建出携带PspA不同家族亚类优势抗原(PspA-Fam1-Clade1和PspA-Fam2-Clade3)的减毒沙门菌为宿主菌的非抗性基因筛选的真核质粒-宿主致死平衡系统,且稳定性好;免疫小鼠鼻咽部抗PspA-Fam1-Clade1和PspA-Fam2-Clade3的特异性sIgA的水平显著高于对照组(P<0.01);免疫小鼠鼻咽部肺炎链球菌定植模型的菌落计数较对照组明显减少(P<0.01)。结论以减毒沙门菌为载体携带PspA不同家族亚类优势抗原是增强肺炎链球菌DNA疫苗鼻咽部黏膜免疫的优化策略。Objective To evaluate the defensive effect of streptococcus pneumoniae DNA vaccine against different invasive pneumococcal,that uses Salmonella-based new carrier system with different family-clade of PspA antigens,and discuss the optimization strategy of streptococcus pneumoniae DNA vaccine in enhancing nasopharyngeal mucosal immunization.Methods Based on the previous research,preparing the recombinant salmonella-based balanced-lethal host-vector system with eukaryotic plasmid encoding PspA-Fam1-Clade1 and PspA-Fam2-Clade3 by molecular cloning technology.Advantage antigens expressions were detected by RT-PCR and Western-blotting.Salmonella with advantage antigens were subculture in an SOB culture medium with or without DAP,and plasmids were extracted and test the stability carrying antigens by PCR.With PBS as contrast,oral immunization BABL/c mice with streptococcus pneumoniae multiple pspA genes DNA vaccine using the Salmonella-based new carrier system,nasopharyngeal sIgA levels were detected by ELISA,and colony counting from different invasive streptococcus pneumonia strains in nasopharyngeal lavage were detected in nasopharyngeal carriage mice model immunized.Result Successfully,salmonella-based balanced-lethal host-vector systems with eukaryotic plasmid encoding PspA-Fam1-Clade1 and PspA-Fam2-Clade3 were builded with good stability carrying antigens.The levels of specifically sIgA against PspA-Fam1-Clade1 and PspA-Fam2-Clade3 in nasopharyngeal lavage of mice immunized significantly higher than that of control group （P 〈 0.01） ; colony count of nasopharyngeal lavage in mouse models immunized were significantly smaller than the control group （P 〈 0.01）.Conclusions Using the Salmonella-based new carrier system carries streptococcus pneumoniae DNA vaccine with multiple pspA genes is a optimization strategy against Streptococcus pneumonia nasopharyngeal colonization for enhancing nasopharyngeal mucosal immune of strcptococcus pneumoniae DNA vaccine.

关 键 词：肺炎链球菌表面蛋白A DNA疫苗 鼻咽部定植 黏膜免疫 减毒沙门菌 

分 类 号：R563.1[医药卫生—呼吸系统]