转化缺陷的R6菌株经黏膜免疫小鼠可抵抗肺炎链球菌有毒株感染  

R6mutant with transformation defect of mucosal immune elicits protection against the infection of Streptococcus pneumoniae in mice

作　　者：徐绣宇[1] 王一平[1] 蔡莺莺[1] 刘宇思[1] 王虹[1] 胥文春[1] 何於娟[1] 张雪梅[1] 尹一兵[1] 

机构地区：[1]重庆医科大学检验医学院检验系临床检验诊断学实验室,重庆400016

出　　处：《临床检验杂志》2013年第4期263-267,共5页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(31070819);重庆市渝中区科技计划项目(20110315);重庆医科大学实验教学管理中心创新实验项目(201117)

摘　　要：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 construct an attenuated strain of Streptococcus pneumoniae （S. pn ）-R6AcomE with transformation defect, and evaluate its protection against the infection of S. pn. Methods Firstly, R6AcomE strain with defected comE gene was construc- ted, and the transformation ability and virulence of R6 and R6AcomE evaluated. Then, BALB/c mice were intranasally immunized with R6 and R6AcomE, respectively, and the titers of specific antibody in serum and the levels of IFN-γ, IL-4 and IL-17A in the su- pernatant of splenocytes detected by ELISA. In the meanwhile, immunized mice were intranasally infected with pneumococcal D39 strain, and their live time and survival rate observed. Results R6AcomE strain with defected comE gene was successfully constructed. The medians of transformation bacterial loads for R6 were 1 970 CFU/mL in vitro and 34 CFU per mice in vivo, respectively, whereas no transformation occurred for R6AcomE both in vitro and in vivo. All mice challenged with 1 v 10s CFU of D39 died in 3 days and those with the same dose of R6 and R6AcomE all survived more than 21 days. Moreover, both R6 and R6AcomE were cleared in 72 hours after infection. After mice were immunized with R6 and R6AcomE, the medians of specific antibody titers in serum were 1.0 × 106 （ P25 = 6.4 × 105, P75 = 1.8 × 106 ） and 1.2 × 106 （P25 = 8.1 × 105, P7s = 1.6 × 106 ） , respectively, which was significantly higher than that in negative controls （ P 〈 0.01 ）. The levels of IL-4 and IL-17A but not IFN-γ from immunized mice with R6 and R6AcomE were significantly higher than that from adjuvant controls （P 〈 0.01 ）. In addition, the mice immunized with R6 and R6AcomE elicited protection against the challenge of D39, and the survival rates were 60% and 70%, respectively. Conclusion The R6AcomE strain, with highly attenuated virulence and no transformation ability, was a safe and effective vaccine candidate, and could induce the protection against S. pn infection.

关 键 词：肺炎链球菌 黏膜免疫 转化缺陷 疫苗 

分 类 号：R378.1[医药卫生—病原生物学]