PspA免疫小鼠抗肺炎链球菌侵袭性感染研究

目的:获取原核表达的肺炎链球菌PspA重组蛋白并研究其作为疫苗的价值. 方法:分离培养肺炎链球菌TIGR4,获取其染色体DNA,采用基因体外重组法将编码PspA抗原表位在内的部分序列克隆到原核表达载体PET-32(a)内,酶切及测序鉴定重组质粒转化到大肠杆菌BL21(DE3)中,经IPTG诱导,将获得的重组蛋白用Western Blot鉴定,镍柱纯化并透析除盐. 用重组蛋白免疫动物,观察PspA抗体对肺炎链球菌感染小鼠的保护作用. 结果:DNA序列与GenBank中的数据相符,所表达纯化的蛋白经Western Blot证实为PspA,其抗体在肺炎链球菌感染中对小鼠有保护作用. 结论:重组PspA刺激产生的抗体能够有效抵抗肺炎链球菌TIGR4侵袭性感染,该重组蛋白可作为肺炎链球菌多肽联合疫苗的组成部分之一.

Protective effect of PspA against invasive infection of Streptococcus pneumoniae in mice

AIM: To obtain purified PspA by prokaryotic expression system and to investigate the effect of PspA antibody on invasive infection of Streptococcus pneumoniae. METHODS: Template DNA was isolated from cultured TIGR4. By gene recombination technology in vitro, the sequence encoding PspA antigen epitope was cloned into PET-32(a) expression vector. After being confirmed by sequencing, expressed antigen protein was identified by Western Blot and purified through Ni~2+ affinity chromatography and processed by dialysis and preserved by freezing and drying. After pneumococcal challenge in actively and passively immunized mice, we compared their median survival times by Mann-Whitney U test. RESULTS: The DNA sequence of the cloned PspA gene was consistent with GenBank data, and PspA fusion protein was proved by Western Blot. PspA can exert a protection against invasive pneumococcal infection. CONCLUSION: A highly expressed recombinant PspA protein has been successfully obtained and proved to exert the protection against invasive pneumococcal infection, suggesting PspA can be a promising candidate vaccine.