Zhang De-guang, Wang Zheng-min, Xu Jiang-hong, Chen Bing, Dai Wen-jia, Fan Xiao-yong
Department of Otorhinolaryngology Head and Neck Surgery, Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, China.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2009 Sep;44(9):762-6.
To study on the preparation of Streptococcus pneumoniae psaA DNA vaccine and to analyse the immunogenicity by the prime-boost strategy.
The psaA gene was amplified from the genome of Streptococcus pneumoniae by PCR, and then was inserted into plasmid pVAX1 and pET28a to construct recombinant expression vectors respectively. 293T cells were transiently transfected with pVAX1-psaA, and RT-PCR analysis of total cell RNA extracts showed successful expression of psaA. BALB/c mices (n = 5) were intramuscularly injected with 100 microg psaA DNA vaccine for three times, and then boosted with 50 microg recombinant PsaA protein. The antibody response against PsaA was measured by ELISA.
The psaA gene was amplified and subcloned successfully. The constructed psaA DNA vaccine was confirmed by DNA sequencing, and the recombinant PsaA protein was purified by the one-step Ni(2+) affinity chromatography. Expression of the PsaA was observed in cells transfected with pVAX1-psaA. The animal experiment results showed that the anti-PsaA level of the DNA prime-protein boosting mice was higher significantly than the other groups (t = 87.518, P < 0.05).
The psaA DNA vaccine was prepared successfully, and the immunogenicity of Streptococcus pneumoniae psaA DNA vaccine could be improved significantly by the DNA prime and protein boost strategy.
研究肺炎链球菌psaA DNA疫苗的制备，并通过初免-加强策略分析其免疫原性。
通过PCR从肺炎链球菌基因组中扩增psaA基因，然后分别插入质粒pVAX1和pET28a构建重组表达载体。用pVAX1-psaA瞬时转染293T细胞，对细胞总RNA提取物进行RT-PCR分析显示psaA成功表达。将100μg psaA DNA疫苗肌肉注射给BALB/c小鼠（n = 5）3次，然后用50μg重组PsaA蛋白进行加强免疫。通过ELISA检测针对PsaA的抗体反应。
成功扩增并亚克隆了psaA基因。通过DNA测序确认构建的psaA DNA疫苗，并用一步Ni(2+)亲和层析法纯化重组PsaA蛋白。在转染pVAX1-psaA的细胞中观察到PsaA的表达。动物实验结果表明，DNA初免-蛋白加强免疫的小鼠抗PsaA水平显著高于其他组（t = 87.518，P < 0.05）。
成功制备了psaA DNA疫苗，DNA初免和蛋白加强策略可显著提高肺炎链球菌psaA DNA疫苗的免疫原性。
