肺炎链球菌黏附素A基因疫苗的构建及对小鼠鼻咽部肺炎链球菌携带的防御作用

目的 探讨肺炎链球菌表面黏附素A(PsaA)基因疫苗的免疫原性及其对小鼠鼻咽部肺炎链球菌携带的防御作用.方法 PCR技术从肺炎链球菌R6标准株扩增PsaA基因,构建重组真核表达质粒pcDNA3.1-PsaA,脂质体法转染BHK-21细胞,逆转录.聚合酶链反应(RT-PCR)及免疫印迹(Western blot)检测重组质粒在BHK-21细胞中的转录和表达.应用PsaA基因疫苗肌肉注射免疫BABL/c小鼠,ELISA检测脾细胞上清IFN-γ分泌,特异性抗体IgG水平及其亚型分布(IgG1/IgG2a).利用BABL/c小鼠肺炎链球菌D39株鼻咽部携带模型,鼻咽部灌洗液菌落计数观察免疫小鼠鼻咽部肺炎链球菌携带改变.结果 RT-PCR及Western blot显示重组PsaA蛋白在BHK-21细胞瞬时表达,ELISA检测到免疫小鼠血清高水平的IFN-γ及特异性IgG抗体(IsG1/IgG2a＜1),小鼠攻击实验证明PsaA基因疫苗免疫组小鼠D39携带菌落计数明显少于对照组(P＜0.01).结论 肺炎链球菌表面毒力蛋白PsaA基因疫苗免疫小鼠激发了抗原特异性的细胞及体液免疫反应,显著减少了小鼠鼻咽部肺炎链球菌的携带,为肺炎链球菌DNA疫苗理想侯选抗原之一.

Construction of DNA vaccine of pcDNA3.1-PsaA and its effects against nasopharyngeal colonization of Streptococcus pneumoniae

Objective To investigate the immunogenicity of DNA vaccine expressing pneumococcal surface adhesion A(PsaA),and its effect against nasopharyngeal colonization of Streptococcus pneumoniae in BALB/c mice.Methods The genomic DNA of the standard Streptococcus pneumoniae strain D39 was isolated.PsaA gene open reading fragment was amplified by polymerase chain reaction(PCR) and cloned into the eukaryotic expression vector pcDNA3.1(+).Recombinant pcDNA3.1-PsaA was transfected into BHK-21 cells using LipofectamineT...