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目的研制一种无转化能力的肺炎链球菌(S.pn)减毒株R6ΔcomE,评价其作为S.pn活菌疫苗的保护效果。方法构建comE基因缺陷的R6ΔcomE株,评价R6、R6ΔcomE株的转化能力及其毒力;用R6、R6ΔcomE株分别对BALB/c小鼠进行黏膜免疫,用ELISA法检测免疫小鼠血清特异性抗体效价和脾细胞上清中IFN-γ、IL-4及IL-17A水平;用D39株经鼻腔黏膜感染免疫小鼠,观察生存时间和生存率。结果成功构建comE缺陷的R6ΔcomE株。体外转化实验中R6株转化菌量中位数为1 970 CFU/mL,体内转化实验中每只小鼠34 CFU,体内外实验中R6ΔcomE株转化菌量均为0;感染1×108CFU D39株的小鼠于3 d内全部死亡,而感染相同剂量R6、R6ΔcomE株的小鼠均存活>21 d,且均于感染后72 h内清除细菌。R6、R6ΔcomE株黏膜免疫小鼠后,血清抗体滴度的中位数(P25,P75)分别达1.0×106(6.4×105,1.8×106)、1.2×106(8.1×105,1.6×106),与相应阴性对照组比较差异有统计学意义(P均<0.01);与相应阴性对照组比较,R6、R6ΔcomE株免疫组IL-4、IL-17A均升高(P均<0.01),而IFN-γ水平差异无统计学意义(P>0.05)。黏膜免疫R6、R6ΔcomE株的BALB/c小鼠可抵抗D39株感染,生存率分别为60%和70%。结论 R6ΔcomE株毒力弱,无转化能力,可诱导对S.pn感染的免疫保护,是一株安全有潜力的S.pn减毒活菌疫苗。
Objective To develop an unreportable S. pneumoniae (S. pn) attenuated strain R6ΔcomE and evaluate its protective effect against S.pn viable vaccine. Methods The R6ΔcomE strain with comE gene defect was constructed to evaluate the transformation ability and virulence of R6 and R6ΔcomE strains. BALB / c mice were immunized with R6 and R6ΔcomE strains respectively. Serum-specific antibody was detected by ELISA The levels of IFN-γ, IL-4 and IL-17A in the supernatant of spleen cells were measured. The mice were immunized with the D39 strain via nasal mucosa to observe the survival time and survival rate. Results The comE-deficient R6ΔcomE strain was successfully constructed. In vitro transformation experiment R6 strain transformable bacteria in the median of 1 970 CFU / mL, in vivo transformation experiment in each mouse 34 CFU, in vitro and in vivo experiments R6ΔcomE strains transformed bacteria were 0; infected 1 × 108CFU D39 strain The mice all died within 3 days, while the mice infected with the R6 and R6ΔcomE strains all survived for> 21 days and all cleared the bacteria within 72 hours after infection. The median antibody titers (P25, P75) of R6 and R6ΔcomE strains were 1.0 × 106 (6.4 × 105, 1.8 × 106) and 1.2 × 106 (8.1 × 105, 1.6 × 106) (P <0.01). Compared with the corresponding negative control group, the levels of IL-4 and IL-17A in the R6 and R6ΔcomE groups were significantly increased (P <0.01), but not in the corresponding negative control group There was no significant difference in IFN-γ levels (P> 0.05). Mucosal immunization BALB / c mice from R6 and R6ΔcomE strains were resistant to D39 infection with survival rates of 60% and 70%, respectively. Conclusions R6ΔcomE strain is a safe and potential strain of attenuated S.pn live attenuated vaccine with low virulence and no transformation ability, which can induce immune protection against S.pn infection.