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

发布时间：2018-05-19 03:05

本文选题：肺炎链球菌蛋白疫苗 + 肺炎链球菌内肽酶O　；参考：《中国当代儿科杂志》2017年05期

【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(P0.05),但均高于PsaA组(P0.05)。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(P0.05),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(P0.05)。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(P0.05),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(P0.05)。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 express prokaryotic expression of Streptococcus pneumoniae endopeptidase Op Pepo and Streptococcus pneumoniae surface adhesin A PsaAn, and to evaluate its protective effect as a vaccine candidate protein. Methods the specific primers were designed and amplified by PCR to construct the recombinant plasmid pET28a, which was transformed into Escherichia coli BL21DE3. After purification, high purity PepO and psaA were obtained. BALB/c mice were immunized with their specific antiserum. The titer of antibody was detected by Elisa. Western blot analysis was used to verify the specificity of the target protein antiserum. BALB/c mice were randomly divided into PsaA group (negative control group) and PepO PsaA combined immunization group (18 mice in each group). Different serotypes of Streptococcus pneumoniae infection models were established to evaluate the immune protection effect of single and combined use of the target protein. Results PepO and PsaA were successfully prepared and obtained. The specificity of the antiserum was verified by Western blot. There was no significant difference in IgA in saliva and IgG titer in serum between PepO group and combined immunized group, but they were higher than that in PsaA group. The protective rate of P0.05A group and combined immunization group on D39 and CMCC31436 mice infected with Streptococcus pneumoniae in nasal cavity was higher than that in negative control group (P 0.05), and the protective rate of PepO group and combined immunization group on D39 mice infected with Streptococcus pneumoniae in nasal cavity group was higher than that in PsaA group. The results of anti-colonization experiment showed that, Compared with the control group, PsaA group and combined immunization group could effectively reduce the colonization of streptococcus pneumoniae CMCC31693 and CMCC31207 in nasopharynx and lung of mice, and the combined immunization group had better effect on reducing the colonization of streptococcus pneumoniae CMCC31207 in the lungs of mice. Conclusion compared with the mice immunized by mucosal pathway, PepO PsaA combined vaccine has better protective effect and can effectively resist the colonization of Streptococcus pneumoniae in nasopharynx and lung.
【作者单位】：四川大学华西第二医院检验科/妇儿疾病与出生缺陷教育部重点实验室;
【基金】：国家自然科学基金(81301399)
【分类号】：R392

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