肺炎球菌荚膜多糖和溶血素结合疫苗的制备及其免疫原性的研究  

The experimental research of tne preparation for pneumococcal conjugate vaccine using pneumolysin as a protein carrier and its immumogenicity in mice

作　　者：陈泽宇[1] 陈兵[1] 王正敏[1] 戴文佳[1] 邓月[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院耳鼻喉科,上海200031

出　　处：《中华微生物学和免疫学杂志》2006年第10期906-910,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(30471872);教育部211工程建设项目

摘　　要：目的研制肺炎球菌荚膜多糖(PS)与肺炎球菌溶血素(蛋白)偶联结合疫苗。方法用PCR扩增肺炎球菌溶血素(Pn)基因,将其克隆入表达载体pET-28a质粒中,然后转化大肠杆菌JM109(DE3)宿主细胞,并以IPTG诱导进行蛋白质表达,对所得蛋白质用固相Ni-NTA树脂作纯化,再用SDS-PAGE鉴定表达的纯化蛋白(rPn)。以福尔马林脱毒rPn,去除其溶血活性,然后用氨基还原法将脱毒的rPn与PS(19F血清型)偶联结合,加入铝佐剂制备成疫苗。以该疫苗腹腔注射接种小鼠,用ELISA法检测小鼠血清中抗PS及抗Pn的抗体水平。结果成功扩增和克隆了Pn基因,经诱导表达得到rPn蛋白,纯化后纯度达90％以上。PS与Pn结合物经凝胶层析柱分离,其洗脱峰较PS与Pn二峰明显前移。用该结合疫苗免疫的小鼠血清中抗PS(19F)抗体及抗Pn抗体的效价均明显高于阴性对照组(P＜0．01)。结论用基因工程技术制备的肺炎球菌溶血素作为蛋白载体,脱毒后与肺炎球菌多糖偶联而成的结合疫苗能在小鼠体内诱导出明显的免疫应答反应。Objective To prepare pnearnococcal conjugate vaccine using pnearnolysin as a protein carrier and evaluate its immunogenicity in mice. Methods The DNA fragment encoding for pneumolysin was amplified by PCR and cloned into plasmid pET-28a, and then the recombinant DNA was transfered to host cell E. coli JM109 （DE3）. The recombinant pneumolysin （rPn） was expressed in vivo and purified by Ni-NTA. The SDS- PAGE was used to identify the rPn. This protein was detoxified by 0.2% or 0.5% formahn. Pnetanococcal polysaccharide （PS, 19F serotype） was conjugated with detoxified rPn using amino reductive method. The conjugate was mixed with the adjuvant Al2 （ OH）3 before vaccination. The antibody response against PS or Pn was measured by ELISA. Results The inserted pneumolysin gene sequence was confirmed by DNA sequencing and the pneumolysin protein was successfully expressed. The relative molecular mass of the expressed product was 52 × 10^3 . This recombinant pneumolysin was detoxified by 0.2% or 0.5% formalin. The PS （ 19F serotype） was successfully conjugated with rPn. There was a significant difference of antibody titer against PS or Pn between vaccinated and non-vaccinated mice （ P 〈 0.01）. Conclusion recombinant pneumolysin can obtained by genetic engineering technology. The PS and rPn conjugate vaccine produced by amino reductive method induced specific immune response in mice.

关 键 词：肺炎球菌 肺炎球菌溶血素 多糖和蛋白结合疫苗 免疫应答 

分 类 号：R392[医药卫生—免疫学]