中国B群流脑菌株不同变异群H因子结合蛋白保护性抗原的筛选

Screening of protective antigen of factor H binding protein of different variants of serogroup B Neisseria meningitidis in China

目的筛选中国流行B群流脑菌株不同变异群的H因子结合蛋白(factor H binding protein,fHbp)的保护性抗原。方法根据中国流行病学数据选择6条中国流行B群流脑菌株不同变异群的fHbp抗原基因序列,优化合成后克隆构建原核表达质粒,转化E.coli BL21(DE3)感受态细胞,表达fHbp蛋白,经镍柱亲和纯化后,免疫BALB/c小鼠,检测其诱导产生的结合抗体和杀菌抗体反应。结果成功构建6个fHbp基因的原核表达质粒,纯化后的fHbp蛋白纯度均超过85%。6个抗原均可诱导产生平均抗体滴度超过1∶40000的结合抗体免疫反应,其中fHbp V2.18、fHbp V3.31和fHbp V3.94抗原可诱导针对fHbp V3.94菌株的杀菌抗体反应,fHbp V2.16和fHbp V2.18抗原可诱导产生针对fHbp V2.16菌株的杀菌抗体反应;确证试验显示,fHbp V2.18和fHbp V3.94抗原均可诱导产生针对fHbp V2.16和f Hbp V3.94菌株的杀菌抗体反应,肌肉免疫诱导产生的杀菌抗体反应高于腹腔免疫,其中fHbp V2.18抗原肌肉免疫诱导的针对f Hbp V2.16和fHbp V3.94菌株的平均杀菌抗体滴度分别为1∶134.4和1∶56,fHbp V3.94抗原肌肉免疫诱导的针对f Hbp V2.16和fHbp V3.94菌株的平均杀菌抗体滴度分别为1∶200和1∶1280。结论通过抗原筛选获得了可诱导杀菌抗体的中国流行B群流脑菌株的fHbp V2和V3抗原各2个,可用于后续B群流脑疫苗的研究。

Objective To screen the protective antigen of factor H binding protein(fHbp)of different variants of serogroup B Neisseria meningitidis prevalent in China.Methods According to the epidemic data,the fHbp gene sequences of different variants of six serogroup B N.meningitides strains prevalent in China were selected,optimized,synthesized and cloned into prokaryotic expression vector.The constructed recombinant plasmid was transformed to competent E.coli BL21(DE3)cells,and the expressed Hbp protein was purified by nickel ion affinity chromatography,with which BALB/c mice were immunized,and the induced binding antibody and bactericidal antibody response were determined.Results Six recombinant plasmids for expression of fHbp gene was successfully constructed.The purity of purified fHbp protein was more than 85%.All the antibody titers induced by the six antigens were more than 1∶40000.The f Hbp V2.18,fHbp V3.31 and f Hbp V3.94 antigens induced bactericidal antibody response against fHbp V3.94 bacterial strain,while fHbp V2.16 and f Hbp V2.18 antigens induced bactericidal antibody response against fHbp V2.16 bacterial strain.Confirmation test showed that both fHbp V2.18 and fHbp V3.94 antigens induced bactericidal antibody responses against fHbp V2.16 and fHbp V3.94 bacterial strains.However,the response level induced by intramuscular route was higher than that by intraperitoneal route.The mean bactericidal antibody titers against fHbp V2.16 and fHbp3.94 bacterial strain induced by f Hbp V2.18 antigen by intramuscular route were 1∶134.4 and 1∶56,while those by fHbp V3.94 antigen were 1∶200 and 1∶1280,respective ly.Conclusion Two fHbp V2 and two fHbp V3 antigens which may induce bactericidal antibody against serogroup N.meningitidis are obtained by antigen screening,which may be used for further study on vaccines.