肺炎球菌表面蛋白A(PspA)的克隆表达及交叉免疫作用

Cloning and Expression of Pneumococcal Surface Protein A and Its Cross-immunoreactivity

摘要:

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20 μg/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。

Abstract:

In this study, we cloned genes coding the N-terminal region of PspA( pneumococcal surface protein A) from five of the most epidemic pneumoniae serotypes in China(FY01, FY05, FY6B, FY19F, FY23F). The obtained genes were respectively constructed into prokaryotic expression vector pET-27b(+), and then recombinant plasmids pET-pspA were respectively transformed into Escherichia coli BL21(DE3) for PspA expression. Induced with lactose, the five recombinant proteins were highly expressed and then purified by Ni-chelation chromatography. PCR with Family special primers was carried out to identify Family of PspA, and Clade of PspA was identified by means of bioinformatics according to the sequencing results. FY01rPspA, FY6B rPspA and FY19FrPspA belonged to Clade 1 of FamilyⅠ, FY05rPspA belonged to Clade 2 from FamilyⅠand FY23F rPspA belonged to Clade 3 from FamilyⅡ. Strong immunogenicity had been demonstrated in mice immuned with FY01rPspA at dose of 20 μg/mL each, of which antiserum antibody titer reached 1:210000. Result of western blotting showed that FY01rPspA antiserm reacted well with Y6B rPspAand FY19FrPspA but poorly with the other two, which suggested that cross-immunoreactivity was restricted in the same Clade. Mice immuned with FY01rPspA protected well against FY6B, while against FY05, FY23F was not effectively protected. The result in this study has much instructive significance for the development of a new efficient vaccine against pneumoniae.