肺炎链球菌表面蛋白 A 的家族Ⅰ不同支系融合蛋白的制备及免疫原性研究  

Preparation of a fusion protein with pneumococcal surface protein A of two different clades of familyⅠand analysis of immunogenicity of the recombinant protein

作　　者：林海英[1,2] 彭永辉[1] 张双玲[1] 罗春华[1] 郑美云[1] 吕唯[1] 詹杜清 

机构地区：[1]福州大学生物科学与工程学院,350100 [2]福州大学药物生物技术与工程研究所

出　　处：《中华微生物学和免疫学杂志》2015年第5期377-381,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金项目（31300755）;福建省自然科学基金（2013J01130）

摘　　要：目的：构建肺炎链球菌家族Ⅰ中支系1和2肺炎链球菌表面蛋白A（ PspA）重组融合蛋白，分析融合蛋白的免疫原性。方法 PCR扩增目的基因片段，构建重组质粒并表达，ELISA检测抗体滴度和亲和度，以及抗蛋白血清特异性和免疫交叉性。结果成功构建重组质粒并原核表达。血清中抗体滴度可达到1×10^4，亲和度达到2×10^5，调理吞噬试验阳性，保护试验效果显著。结论肺炎链球菌家族Ⅰ中支系1和2肺炎链球菌表面蛋白A（ PspA）重组融合蛋白具有较高的免疫原性，能够诱导小鼠产生高滴度和高特异性的抗体，具有免疫交叉保护性。Objective To construct a recombinant fusion protein with pneumococcal surface pro-tein A （PspA） of Stretococcus pneumonia （SPN） familyⅠclade 1 and 2, and to analyze the immunogenici-ty of the fusion protein.Methods The gene fragments encoding theα-helix of PspA of the two clades were amplified by PCR and then inserted into the expression vector pET-27b（＋） to construct the recombinant ex-pression plasmid.The transformed Escherichia coli BL21 strains carrying expression plasmid were induced by IPTG to express the recombinant protein.The titers and affinity of antibodies against PspA protein were measured by ELISA.An opsonophagocytic assay and an animal experiment were performed to evaluate the immunogenicity of the recombinant protein.Results Double enzyme cutting and gene sequencing confirmed the two purpose gene fragments were correctly expressed in the expression vector pET-27b（＋）.The titers of anti-PspA antibody in the serum of Kunming （ KM） mice immunized with the fusion protein were 1 ×10^4 . The affinity of anti-PspA antibody reached to 2×10^5 .The rates of recombinant PspA6B-PspA05 protein me-diated phagocytosis for SPN6B, SPN05 and SPN01 strains were 20%, 15% and 8.8%, respectively.No SPN23F strain was engulfed by macrophages upon the stimulation with PspA6B-PspA05 protein.The survival rates of mice injected with SPN05, SPN6B, SPN01 and SPN23F strains were respectively 75%, 92%, 75%and 33%upon the immunization of PspA6B-PspA05 protein.Conclusion The recombinant fusion protein PspA6B-PspA05, constructed with the PspA proteins of Stretococcus pneumonia familyⅠclade 1 and 2, was successfully expressed in the E.coli prokaryotic system with the advantage of high immunogenicity.High ti-ters of anti-PspA antibodies with high specificity were induced in KM mice upon the stimulation with Ps-pA6B-PspA05 protein.Moreover, a cross-protective immunity was induced in KM mice upon the immuniza-tion with PspA6B-PspA05 protein.

关 键 词：肺炎链球菌 肺炎链球菌表面蛋白A 重组蛋白 免疫原性 

分 类 号：R392[医药卫生—免疫学]