肺炎链球菌表面蛋白A多价DNA疫苗交叉免疫保护作用评价张巧林.DOCVIP

肺炎链球菌表面蛋白A多价DNA疫苗交叉免疫保护作用评价 张 巧，林科雄，姚 伟，李 琦，钱桂生，王长征，马千里 （400037 重庆，第三军医大学新桥医院全军呼吸内科研究所，全军呼吸病研究重点实验室） [摘要] 目的 评价肺炎链球菌表面蛋白A（pneumococcal surface protein A，PspA）优势家族亚类抗原的肺炎链球菌多价DNA疫苗抵御不同肺炎链球菌来源的异种PspA分子毒力作用的能力。方法 以包含PspA抗原Fam1的Clade1亚类和Fam2的Clade3亚类DNA序列的T载体为模板， 聚合酶链反应（PCR）扩增具有高保护性抗体效价和强交叉反应的PspA优势家族亚类PspA-Fam1-Clade1和PspA-Fam2－Clade3片段，克隆至真核载体pcDNA3.1(+)，构建重组质粒pcDNA3.1-Clade1和pcDNA3.1-Clade3，免疫印迹（Western-blot）及逆转录聚合酶连反应（RT-PCR）检测重组质粒在哺乳动物细胞BHK-21中的表达。重组肺炎链球菌PspA多价DNA疫苗（pcDNA3.1-Clade1＋pcDNA3.1-Clade3）肌肉注射免疫小鼠，观察免疫小鼠鼻咽部PspA异家族来源的不同肺炎链球菌携带情况改变和腹腔攻击小鼠21d的生存情况。结果 成功构建肺炎链球菌PspA优势家族亚类抗原的肺炎链球菌多价DNA疫苗。疫苗（pcDNA3.1-Clade1＋pcDNA3.1-Clade3）免疫组小鼠鼻咽部携带的PspA异家族来源的不同肺炎链球菌菌落计数无显著性差异（P＞0.05），而明显少于空白质粒及PBS对照组小鼠（P0.01）；疫苗（pcDNA3.1-Clade1＋pcDNA3.1-Clade3）免疫组小鼠针对PspA异家族来源的不同肺炎链球菌腹腔攻击后中位生存时间无显著差异（P＞0.05），而明显长于空白质粒及PBS对照组小鼠（P0.01）。 结论 PspA优势家族亚类PspA-Fam1-Clade1和PspA-Fam2－Clade3的靶抗原组合形式能够有效解决PspA的抗原多样性问题，所构建的肺炎链球菌DNA疫苗有望针对PspA异家族来源的临床肺炎链球菌株感染。 [关键词] 肺炎链球菌；肺炎链球菌表面蛋白A；核酸疫苗 [中图法分类号] R392233; R392. 7; R563. 1 [文献标志码] A Evaluation of Cross- protection of Streptococcus pneumoniae DNA vaccine with pspA gene against Streptococcus pneumoniae with different PspA Family clades Zhang Qiao , Lin Kexiong, Yao Wei, Li Qi, Qian Guisheng,Wang Changzheng, Ma Qianli（ Department of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University. Chongqing, 400037, China） Corresponding Author: MA Qian-li, E-mail: cqmql@ 163.com [Abstract] Objective To evaluate the ability of Streptococcus pneumoniae multivalent DNA vaccine with dominant pneumococcal surface protein A（pspA） clades genes against Streptococcus pneumoniae with different PspA family clades. Methods Polymerase chain reaction (PCR) amplified the PspA -Fam1-Clade1 and PspA-Fam2-Clade3 fragments from the recombinant T vectors. Which were antigens with a high protective antibody titers and strong cross-reaction against the other PspA family clades. And then they were cloned into the eukaryotic vector pcDNA3.1 (+) for th