肺炎链球菌内肽酶O及表面黏附素A联合蛋白疫苗免疫保护作用研究

Immunoprotective effect of combined pneumococcal endopeptidase O and pneumococcal surface adhesin A vaccines against Streptococcus pneumoniae infection

摘要:

目的 原核表达肺炎链球菌内肽酶O（PepO）及肺炎链球菌表面黏附素A（PsaA），评价其作为疫苗候选蛋白的免疫保护效果。方法 设计pepO，psaA目的基因片段的特异性引物，经PCR扩增，构建重组质粒pET28a（+）-pepO，pET28a（+）-psaA，重组质粒转化大肠埃希菌BL21（DE3）诱导表达，经纯化后获得高纯度目的蛋白PepO及PsaA。经黏膜免疫BALB/c小鼠制备其特异性抗血清，ELISA检测抗体效价，Western blot分析验证目的蛋白抗血清的特异性。将BALB/c小鼠随机分为阴性对照组、PepO组、PsaA组及PepO+PsaA联合免疫组，每组18只。构建不同血清型肺炎链球菌感染模型，评估目的蛋白单用及联合使用的免疫保护效果。结果 成功制备并获得目的蛋白PepO及PsaA，经Western blot验证目的蛋白抗血清特异性好。PepO组和联合免疫组小鼠唾液中IgA及血清中IgG效价比较差异无统计学意义（P > 0.05），但均高于PsaA组（P < 0.05）。PepO组、PsaA组及联合免疫组对鼻腔感染肺炎链球菌D39及CMCC31436小鼠的保护率高于阴性对照组（P < 0.05），且PepO组及联合免疫组对鼻腔感染肺炎链球菌D39小鼠的保护率高于PsaA组（P < 0.05）。抗定植实验结果显示，与对照组比较，PepO组、PsaA组及联合免疫组均能有效降低肺炎链球菌CMCC31693及CMCC31207在小鼠鼻咽部和肺部的定植（P < 0.05），且联合免疫组对降低肺炎链球菌CMCC31207在小鼠肺部的定植效果更好（P < 0.05）。结论 PepO+PsaA联合疫苗经黏膜途径免疫小鼠后，较单用对小鼠有更好的保护作用，能有效抵抗肺炎链球菌在鼻咽部、肺部的定植，是一组较有开发潜力的蛋白疫苗。

Abstract:

Objective To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins. Methods Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a (+)-pepO and pET28a (+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination. Results The target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P > 0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P < 0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P < 0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P < 0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P < 0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups. Conclusions Combined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.