A族链球菌表面新发现蛋白Fba真核表达质粒的构建及其诱导的免疫应答

Construction of eukaryotic expression plasmid encoding Fba protein of group A streptococcus and elicitation of immune responses by it

目的:构建新发现的A族链球菌(GAS)表面蛋白Fba的真核表达质粒,并将其以及Fba蛋白、M蛋白分别免疫小鼠,对它们诱导的体液免疫及细胞免疫应答进行评价分析。探讨Fba蛋白作为GAS候选疫苗的可能性。方法:以SSI-9菌株(GASM1血清型标准株)作为模板,采用PCR方法扩增Fba基因,测序正确后克隆至真核表达质粒pcDNA3.1,构建真核表达质粒pcDNA3.1/fba;将雌性CD1小鼠随机分成6组,分别为Fba蛋白免疫组、M蛋白免疫组、pcDNA3.1/fba+Fba蛋白免疫组、pcDNA3.1/fba免疫组、pcDNA3.1空质粒对照及PBS对照组。ELISA检测各免疫组血清IgG的动态变化水平;脾细胞增殖试验检细胞增殖水平,并用流式细胞术检测小鼠体内CD4+、CD8+淋巴细胞的变化。试验结果经SPSS10.0统计处理。结果:成功构建了真核表达质粒pcDNA3.1/fba;质粒或蛋白免疫后小鼠IgG产生水平以Fba蛋白免疫组增高最为明显,其次为Fba质粒蛋白混合免疫组及Fba质粒组。特异性抗原诱导后体外脾细胞增殖试验显示:pcDNA3.1/fba免疫组增殖水平明显高于其它组,流式细胞检测结果与此一致,并显示以CD4+T细胞水平升高为主,CD8+T细胞水平在各组别之间也有一定的差异。结论:(1)Fba蛋白与M蛋白一样均能有效地诱导机体产生抗体,提示Fba蛋白很有希望成为GAS的候选疫苗。(2)成功构建了Fba蛋白的真核表达质粒pcDNA3.1/fba,且该重组质粒能有效地诱导抗链球菌所需的抗体,并能增强CD4+T细胞、CD8+T细胞的增殖功能。

Objective：To construct eukaryotic expression plasmid ecording a novel surface protein Fba of GAS, and to explore its impact on host immune responses. Methods：Fba gene was amplified by PCR using strain SSI-9 （GAS M1 serotype isolates） as the template, then cloned into pcDNA3.1 for constructing eukaryotic expression plasmid pcDNA3.1/fba and sequenced. Female CD1 mice were randomly individed into 6 groups, and immunized respectively with Fba protein, M protein, pcDNA3.1/fba ＋ Fba protein, pcD- NA3.1/fba, pcDNA3.1 and PBS as control. Blood was obtained from the mice and specific antibody of IgG was detected by ELISA. Spleen cells were assessed with lymphocyte proliferation assays. CD4^＋ T cell and CD8^＋ T cell were detected by flow cytometry （FCM）. Assay results were analyzed with SPSS10.0. Results：The IgG against Fba protein kept hightest levels in group immunized with Fba protein. The levels of lymphocyte proliferation, CD4^＋, CD8^＋ T cell were significantly high in the group pcDNA3.1/fba. Conclusion： （ 1 ） Just as M protein, the antibody to Fba protein could be efficiently induced by immunization with Fba protein, which showed that Fba protein was hopefully to be a candidate protein for vaccine against GAS. （2） Eukaryotic expression plasmid pcDNA3.1/fba was successfully constructed and this recombinant plasmid could efficiently induce antibody and CD4^＋ T cell for againsting GAS.