A族链球菌多价疫苗的完善制备及其动物免疫保护作用研究

Optimization and evaluation of multi-epitopes vaccine and its immune protection against to GAS infection

目的:完善A族链球菌(GAS)多价疫苗并探究其免疫保护效果。方法:构建GAS的多肽疫苗F7M6,将其中的M6多肽克隆作为对照,表达纯化后,将两种蛋白连同本室保存的GAS M1蛋白分别免疫小鼠,末次免疫后10 d,半数小鼠行ELISPOT检测IL-4和IFN-γ,ELISA检测各组的Ig G、Ig G1、Ig G2a;同时,以F7M6蛋白为诊断抗原检测其与抗链"O"阳性人血清的特异性结合;致死量的M1GAS攻毒并计算存活率。结果:无论是诱导细胞免疫,还是诱导抗体产生,F7M6组的水平均为最高;攻毒后F7M6蛋白的生存率为66.7%。F7M6蛋白检测抗链"O"阳性血清的阳性检出率为95%。结论:GAS的多肽疫苗F7M6能诱导特异性细胞和体液免疫应答,对GAS感染具有良好的保护效果,且F7M6的多价表位涵盖于时下流行的大部分GAS的血清型中。

Objective： To optimize group A streptococcus（ GAS） polyvalent epitope-based vaccine,and explore its immune protection effect. Methods： Psolyvalent vaccine of GAS,F7M6,was constructed and expressed,in which the M6 peptide was constructed as control. 12 mice of each group were immunized with 20 μg per dose of purified F7M6,M6 or M protein（ stored in our lab）,and PBS as negative control. Half of the mice in each group were sacrificed on 10 days of the last immunization,and the levels of Ig G,Ig G1 and Ig G2 a,were detected by ELISA and the levels of IL-4 and IFN-γ were detected by ELISPOT. At the same time,the left immunized animals were challenged with a lethal dose of M1 GAS,and determined the protective effect of the vaccine. Besides,the specific binding ability of F7M6 protein with the sera from ASO-positive patients was detected by ELISA. Results： In F7M6 group,the levels of not only sera Ig G,Ig G1,Ig G2 a,but also IL-4 and IFN-γ were the highest one compared with other groups. Following challenge with type M1 GAS,the survival rates of F7M6 group was 66. 7%. Additionally,when F7M6 acted as diagnostic antigen,the positive detective rate of ASO-positive patient sera was 95%. Conclusion： Polypeptides F7M6,could elicit the best humoral and cell-mediated immune response than other groups,and induce a protective effect in immunized mice against M1 GAS infection. Excitedly,the epitopes of F7M6 protein covers the most of GAS serotypes at present.