脑膜炎球菌表面蛋白fHBP与PorA的融合表达及其免疫原性  

Fusion expression and immunogenicity of Neisseria meningitidis surface proteins fHBP and PorA

作　　者：吴根鹏[1] 袁萍[1] 王月红[1] 王晓[1] 毕慧[1] 黄帼英[1] 徐帆洪[1] 朱为[1] 

机构地区：[1]上海生物制品研究所有限责任公司第三研究室,上海200051

出　　处：《中国生物制品学杂志》2016年第2期124-128,共5页Chinese Journal of Biologicals

摘　　要：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 recombinant plasmid for fusion expression of Neisseria meningitidis （Nm） surface proteins fHBP and PorA, and determine the immunogenicity of fHBP antigen and fHBP-PorA fusion antigen. Methods The fHBP and PorA gene sequences were screened and optimized, and linked to the C-terminus of fHBP by the designed linker. The constructed recombinant plasmids pET-24b-fHBP and pET-24b-fHBP-PorA were transformed to competent E. coli BL21 （DE3） for expression under induction of IPTG. The expressed protein was purified by nickel ion affinity chromatography and gel filtration chromatography, with which rabbits were immunized. The prepared antisera were evalu- ated for immunogenicity by indirect ELISA, flow cytometry and serum bactericidal assay （SBA）. Results Recombinant plasmids pET-24b-fHBP and pET-24b-fHBP-PorA were constructed correctly as proved by restriction analysis and sequencing. Recombinant fHBP was expressed in a soluble form, while fHBP-PorA in a form of inclusion body. All the titers of antisera were more than 1.0 × 105. Flow eytometry showed specific binding of antisera with Nm. However, the antisera against fHBP-PorA showed a certain cross reaction with various Nm strains. Both the bactericidal titers of antisera against fHBP and fHBP-PorA to Nm29019 strain were 1 ： 128. Conclusion The fHBP showed good antigenicity and immuno- genicity, which might be a good candidate for development of vaccines against B group Nm. However, the multiple anti- gen fusion strategy should be further optimized.

关 键 词：脑膜炎球菌 fHBP蛋白 PorA蛋白 融合表达 免疫原性 

分 类 号：R378.15[医药卫生—病原生物学] R392-33[医药卫生—基础医学]