肺炎链球菌毒力蛋白DNA疫苗优势抗原组合筛选及鉴定.docVIP

肺炎链球菌毒力蛋白DNA疫苗优势抗原组合筛选及鉴定 马千里，张 巧，姚 伟，李 琦，王长征（第三军医大学第二附属医院全军呼吸内科研究所第三军医大学新桥医院全军呼吸内科研究所，重庆400037） 提 摘 要： 目的 探讨肺炎链球菌DNA疫苗候选抗原联合免疫的优势抗原组合方式。方法 分子克隆肺炎链球菌表面蛋白A(PspA)N端及肺炎链球菌溶血素（Ply）基因序列，构建重组质粒pcDNA3.1-PspA’和pcDNA3.1-PBD（PBD为Ply点突变减毒体），免疫印迹（Western－ blot）检测重组质粒在哺乳动物BHK-21细胞中的表达（包括前期工作中构建的重组质粒pcDNA3.1-PsaA）。将三3种重组基因疫苗以不同方式组合肌肉注射免疫BABL/c小鼠（B组：pcDNA3.1-PsaA+pcDNA3.1-PspA’, C组：pcDNA3.1-PsaA’+pcDNA3.1-PBD, D组：pcDNA3.1-PspA + pcDNA3.1-PBD，E组：pcDNA3.1-PsaA + pcDNA3.1-PspA’+ pcDNA3.1-PBD），检测各组小鼠血清特异性抗体IgG水平，观察免疫小鼠鼻咽部肺炎链球菌D39携带改变和D39腹腔攻击小鼠21天生存情况d生存情况。结果 成功构建3种重组真核表达载体，ELISA检测发现检测结果显示B组小鼠血清特异性IgG抗体水平与组E组相似（P＞0.05），均明显高于A、组C和组、D及A组（[A组：对照空质粒组pcDNA3.1(+)）]小鼠（P0.01）；免疫小鼠鼻咽部D39携带菌落计数提示B组小鼠相似于E组E小鼠（P＞0.05），并明显少于组C，、 组D组和和阴性对照组（A组和PBS组，P0.01），并且D39腹腔攻击小鼠21天生存时间分析亦提示其中位生存时间明显长于d生存时间分析亦提示其中位生存时间明显长于C组组和D组及阴性对照组（B组和PBS组）（P0.01）。结论 PspA’联合PsaA的抗原组合方式具有高效的协同保护效能，可作为肺炎链球菌DNA疫苗的优势候选抗原组合。 关键词 肺炎链球菌；肺炎链球菌表面黏附素A；肺炎链球菌表面蛋白A；肺炎链球菌溶菌酶；基因疫苗 Screening of dominant antigen formats of DNA vaccine ofagainst Streptococcus pneumoniae and researchers of their protections MA Qian-li，, ZHANG Qiao，, YaoAO Wei，, LI Qi，, WANG Chang-zheng (Institut e of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China) Abstract: Objective To screen the different formats of dominant antigens formats of DNA vaccine of Streptococcus pneumoniae. Methods The fragments of pneumococcal surface protein A (PspA’) and PBD （[mutant of pneumococcal lysozyme (Ply)]） from Streptococcus pneumoniae R6 genome were amplified and then cloned into the eukaryotic expression vector pcDNA3.1(+). These recombinant vectors pcDNA3.1-PspA’, pcDNA3.1-PBD and pcDNA3.1-PsaA (pneumococcal surface adhesin A, (producted in our preliminary work) were transfected into BHK-21 cells, and their expressions were detected with Western- blotting. In 7 d after receiving an injection of cardiotoxin into the quadriceps muscle of thigh, BALB/c mice groups (Group A: pcDNA3.1(+),; Group B: pcDNA3.1-PsaA+pcDNA3.1-PspA’,;