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本工作从中国临床最常见的5种荚膜血清型肺炎链球菌(FY01,FY05,FY6B,FY19F,FY23F)克隆获得编码PspA蛋白N端α-螺旋区的基因片段。分别将5种PspA基因片段克隆到表达载体pET-27b(+)上,成功构建重组质粒pET-pspA,转化大肠杆菌BL21(DE3)。经乳糖诱导表达和亲和层析分离,获得高纯度重组蛋白。通过家族专属引物PCR的方法鉴定这5种荚膜血清型肺炎球菌的PspA蛋白所属家族,并进一步根据基因测序结果通过生物信息学方法鉴定了所属支系。FY01 rPspA、FY6B rPspA、FY19F rPspA同属于家族Ⅰ支系1,FY05 rPspA属于家族Ⅰ支系2,FY23F rPspA属于家族Ⅱ支系3。以FY01 rPspA免疫小鼠,在20μg/mL免疫剂量时抗体滴度达到1:210000,显示了重组蛋白较好的免疫原性。Western blotting交叉免疫反应性研究表明,抗FY01 rPspA抗血清与FY01 rPspA、FY6B rPspA和FY19F rPspA反应性较好,而与另外两种重组蛋白几乎无反应,提示PspA交叉免疫作用仅限于本支系内。动物保护实验表明,FY01 rPspA免疫的小鼠对FY6B、FY01两种菌的攻击具有较好的保护作用,对FY05、FY23F的攻击未见明显的保护作用。本研究成果对于研制高效肺炎球菌疫苗具有重要的指导意义。
In this work, we cloned the gene encoding the N-terminal α-helix region of PspA protein from five species of capsular pneumococci (FY01, FY05, FY6B, FY19F, FY23F) that are the most common in clinical practice in China. Five PspA gene fragments were cloned into the expression vector pET-27b (+) respectively. The recombinant plasmid pET-pspA was successfully constructed and transformed into E. coli BL21 (DE3). Induced by lactose expression and affinity chromatography separation, high purity recombinant protein. Families of PspA proteins belonging to the five capsular serotypes of pneumococci were identified by family-specific primer PCR, and their families were identified by bioinformatics based on the gene sequencing results. FY01 rPspA, FY6B rPspA, FY19F rPspA belong to the same family as I, 1, FY05 rPspA belong to I family, 2, FY23F rPspA belong to family II 3. The mice immunized with FY01 rPspA showed an antibody titer of 1: 210,000 at a dose of 20 μg / mL and showed good immunogenicity of the recombinant protein. The results of Western blotting showed that the anti-FY01 rPspA antiserum had good reactivity with FY01 rPspA, FY6B rPspA and FY19F rPspA, but had little reaction with the other two recombinant proteins, suggesting that the cross-immunization of PspA was limited to this branch . Animal protection experiments showed that mice immunized with FY01 rPspA had a good protective effect against both FY6B and FY01 and no obvious protective effect against FY05 and FY23F. The results of this study for the development of highly effective pneumococcal vaccine has an important guiding significance.