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目的构建脑膜炎球菌(Neisseria meningitidis,Nm)表面蛋白fHBP重组表达质粒,并与PorA蛋白融合表达,对单抗原fHBP和融合抗原fHBP-PorA的免疫原性进行检测。方法筛选并优化fHBP和PorA基因序列,设计连接片段将PorA连接至fHBPC-端,构建重组表达质粒pET-24b-fHBP和pET-24b-fHBP-PorA,分别转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达。表达的重组蛋白经镍柱亲和层析和凝胶过滤层析后免疫家兔,制备抗血清,采用间接ELISA、流式细胞术和微量血清杀菌力试验(serum bactericidal assay,SBA)评价抗原的免疫原性。结果经双酶切和测序鉴定,两个蛋白的重组表达质粒构建正确。重组蛋白fHBP以可溶形式表达,fHBP-PorA以包涵体形式表达,纯化后纯度均在90%以上。抗血清滴度均在1.0×10~5以上。流式细胞术检测显示,抗血清与Nm可发生特异性结合,且fHBP-Por A抗血清显示出一定的交叉反应。fHBP和fHBP-PorA抗血清对Nm29019的杀菌滴度为1∶128。结论 fHBP具有较好的抗原性和免疫原性,具备B群脑膜炎疫苗开发前景。多抗原融合策略需更多的优化考虑。
Objective To construct the fHBP recombinant expression plasmid of Neisseria meningitidis (Nm) surface protein and to fuse with PorA protein to test the immunogenicity of single antigen fHBP and fusion antigen fHBP-PorA. Methods The fHBP and PorA gene sequences were screened and optimized. The adapter fragment was designed to connect PorA to fHBPC-terminus. The recombinant expression plasmids pET-24b-fHBP and pET-24b-fHBP-PorA were constructed and transformed into competent E. coli BL21 (DE3) Cells, induced by IPTG. The expressed recombinant protein was immunized to rabbits by affinity chromatography and gel filtration chromatography. The antiserum was prepared and the expression of antigen was evaluated by indirect ELISA, flow cytometry and serum bactericidal assay (SBA) Immunogenicity. Results Double enzyme digestion and sequencing showed that the two recombinant plasmids were constructed correctly. The recombinant protein fHBP was expressed in soluble form and fHBP-PorA was expressed in inclusion form. The purity of fHBP-PorA was over 90% after purification. Antiserum titers were 1.0 × 10 ~ 5 or more. Flow cytometry showed that the antiserum could specifically bind to Nm, and the fHBP-Por A antiserum showed some cross-reaction. Bactericidal titers of fHBP and fHBP-PorA antisera against Nm29019 were 1: 128. Conclusion fHBP has good antigenicity and immunogenicity, and possesses the prospect of development of meningitis group B vaccine. Multi-antigen fusion strategy needs more optimization considerations.