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目的探讨肺炎链球菌146位氨基酸缺失的溶血素ΔA146Ply蛋白的保护作用,评价其作为肺炎链球菌疫苗候选蛋白的可行性。方法将BALB/c小鼠分为2组:试验组(15μgΔA146Ply蛋白与1 mg/ml CT佐剂的混合物30μl)和对照组(PBS与CT佐剂的混合物30μl),经鼻腔免疫小鼠,每周1次,连续4周。末次免疫1周后,ELISA检测免疫小鼠血清及唾液中特异性抗体水平;并进行ΔA146Ply对多种血清型肺炎链球菌感染的主动保护试验及其特异性抗血清的被动保护试验;West-ern blot分析Ply蛋白在肺炎链球菌中的保守性;间接ELISA检测免疫小鼠血清IgG抗体亚型和细胞因子IL-17A水平。结果试验组小鼠血清中IgG效价为5.12×105,唾液中IgG及IgA效价分别为4.0×103和4.8×103;ΔA146Ply黏膜免疫可有效延长小鼠的生存时间,并提高其生存率;其对肺炎链球菌NCTC7466、CMCC(B)31436、CMCC(B)31614和CMCC(B)31207的保护率分别为50.0%、41.7%、50.0%和41.7%;被动免疫保护试验试验组小鼠的存活率为70%;ΔA146Ply黏膜免疫诱导的抗体亚型主要为IgG1和IgG2b,IL-17A的水平高达(678.55±189)pg/ml,于刺激后48 h达峰值。Ply在4种菌株中均具有良好的保守性。结论ΔA146Ply黏膜免疫可诱导小鼠产生体液免疫和细胞免疫反应,能有效抵抗多株致病性肺炎链球菌的感染,提示ΔA146Ply是肺炎链球菌较好的疫苗候选蛋白。
Objective To investigate the protective effect of amino acid missing hemolysin ΔA146Ply from Streptococcus pneumoniae 146 and to evaluate its feasibility as a candidate protein of Streptococcus pneumoniae vaccine. Methods BALB / c mice were divided into two groups: mice in the experimental group (30 μl of a mixture of 15 μg ΔA146Ply protein and 1 mg / ml CT adjuvant) and a control group (30 μl of a mixture of PBS and CT adjuvant) 1 week, 4 weeks in a row. One week after the last immunization, the levels of specific antibodies in the serum and saliva of the immunized mice were detected by ELISA; and active protection tests of ΔA146Ply against various serotypes of S. pneumoniae and passive protection tests of specific antisera were carried out. West-ern blot analysis of Ply protein in Streptococcus pneumoniae conservative; indirect ELISA detection of serum IgG antibody subtype and cytokine IL-17A levels in mice. Results The titer of IgG and IgA in the saliva was 4.0 × 103 and 4.8 × 103, respectively. The mucosal immunity of ΔA146Ply could prolong the survival time and increase the survival rate of mice. The protective rates against S. pneumoniae NCTC7466, CMCC (B) 31436, CMCC (B) 31614 and CMCC (B) 31207 were 50.0%, 41.7%, 50.0% and 41.7%, respectively; The survival rate was 70%. The antibody subtypes induced by ΔA146Ply mucosal immunization were mainly IgG1 and IgG2b, and the levels of IL-17A were as high as (678.55 ± 189) pg / ml, reaching the peak at 48 h after stimulation. Ply was well conserved among the four strains. Conclusion ΔA146Ply mucosal immunization can induce humoral and cellular immune responses in mice, which can effectively resist the infection of many strains of pathogenic S. pneumoniae, suggesting that ΔA146Ply is a good vaccine candidate protein of S. pneumoniae.