重组脑膜炎球菌P64K蛋白的表达、纯化及载体作用

Expression, purification and carrier effect of recombinant P64K meningococcal protein

目的表达并纯化重组脑膜炎球菌P64K蛋白，观察其载体作用。方法利用基因工程技术在大肠杆菌中表达P64K蛋白，用硫酸铵盐析沉淀和两步层析纯化P64K蛋白。以重组P64K蛋白为载体蛋白，以已二酰肼为连接剂，在碳化二亚胺的作用下，将P64K蛋白与活化的A群脑膜炎球菌多糖（group A meningococcal polysaccharide, GAMP）结合，制备GAMP-P64K结合物。免疫BALB／c小鼠，间接ELISA法测定血清特异性抗P64K蛋白和GAMPIgG抗体，分析P64K蛋白的免疫原性。结果重组P64K蛋白在大肠杆菌中主要以可溶性形式表达，表达量约占菌体总蛋白的35％。动物实验证明，重组P64K蛋白具有较强的免疫原性，表明P64K蛋白具有载体蛋白作用。结论在大肠杆菌中成功表达了重组P64K蛋白，以重组P64K蛋白为载体制备的GAMP—P64K结合物具有良好的免疫原性。这为以重组P64K蛋白为蛋白载体制备其他结合疫苗奠定了实验基础。

Objective To express and purify the recombinant P64K meningcoccal protein and study its effect as a carrier protein. Methods The P64K meningcoccal protein was expressed in E. coli by gene engi- neering technique and purified by ammonium sulfate salting-out precipitation and two steps of chromatography. Using adipic acid dihydrazide as a linking agent, the purified P64K protein was conjugated to the activated group A meningococcal polysaccharide （GAMP） by carbodimide-mediated coupling. BALB/c mice were immu- nized with the GAMP-P64K conjugate, and the specific anti-P64K protein and anti-GAMP IgG antibodies in sera were detected by indirect ELISA. Results The recombinant P64K protein was expressed mainly in a soluble form in E. coli, accounting for approximately 35% of total somatic protein. The animal experiment showed that the recombinant P64K protein had stronger immunogenicity, indicating its cartier effect. Conclusions The recombinant P64K protein is successfully expressed in E. coli, and the GAMP-P64K conjugate shows good immunogenicity. It lays down an experimental foundation for other conjugate vaccines using P64K as a carrier protein.