ClpP融合蛋白诱导的免疫反应可抵抗不同型别肺炎链球菌的侵袭性感染

目的 ClpP融合蛋白在调节肺炎链球菌其他毒力因子表达和入血过程中起重要作用,评价Trx-ClpP融合蛋白对不同型别肺炎链球菌的侵袭性感染的免疫保护效果,为后续蛋白疫苗的开发提供依据.方法 含有重组质粒pET-32a(+)/ClpP工程菌BL21(DE3)经IPTG诱导后表达Trx-ClpP融合蛋白,纯化后与铝佐剂混合经腹腔注射免疫小鼠,而对照小鼠则用PBS与铝佐剂混合免疫.免疫程序:初次免疫5周后用ELISA法测小鼠针对ClpP融合蛋白的抗体免疫应答水平,末次免疫2周后用12个型别肺炎链球菌攻击,监测其生存时间.结果 重组ClpP融合蛋白主动免疫BALB/c小鼠后,产生了高滴度的anti-ClpP融合蛋白抗体,对多个血清型的肺炎链球菌菌血症小鼠模型的保护效果较对照组具有统计学意义.结论 ClpP融合蛋白免疫小鼠后可抵抗1、2、3、4、5、6B、7F、9V、14、18C、19F、23F等12个型别的肺炎链球菌侵袭性感染,提示以ClpP融合蛋白为活性成分的疫苗具有较高的开发价值.

Protective effect of ClpP fusion protein against invasive infection of different serotype Streptococcus pneumoniae in routine models

Objective To evaluate the protective effects elicited by ClpP fusion protein in animal protection tests. Methods Pneumococcal antigens were purified from recombinant Escherichia coli express-ing CIpP cloned gene. 6- to 8-week-old female BALB/c mice were immunized intraperitoneally with either ClpP or PBS plus alum. Every mouse received three doses of 20 μg antigen or PBS in 100 mg of alum adju-vant at 14 d intervals. Sera were collected from mice and analyzed by ELISA 1 week after the third immuni-zation, Intraperitoneal-challenge experiments with 12 different serotypes of Streptococcus pneumoniae were carried out 2 weeks after the third immunization, and we compared their median survival times and survival rates respectively by Mann Whitney U test and Fisher exact test. Results ELISA analysis demonstrated high titer specific antibody responses to ClpP. The median survival times for mice immunized with ClpP pro-tein antigens in adjuvant were significantly longer than those for mice that received the adjuvants alone. Con-clusion A highly expressed recombinant ClpP protein has been successfully obtained and proved to exert the protection against invasive pneumococcal infection without relation of serotype, suggesting ClpP can be a promising candidate vaccine.