B族链球菌表面蛋白C5a肽酶系统及鼻内黏膜免疫的研究

System and intranasal immunization of group B streptococcal CSa peptide

目的研究B族链球菌表面蛋白C5a肽酶（streptococcal C5a peptidase，ScpB）系统及其鼻内途径免疫后在小鼠血清及肺、直肠、阴道等黏膜部位特异性抗体水平，探讨ScpB蛋白黏膜免疫应答的效果。方法在大肠杆菌BL21中表达ScpB蛋白，亲和层析法进行纯化，质谱分析法对所纯化的蛋白进行鉴定。小鼠随机分为5组，每组10只。分别为对照组、皮下30μg组、鼻内5μg组、鼻内10μg组和鼻内30μg组。用ELISA法检测小鼠血清及黏膜萃取液中特异性抗体水平；调理吞噬试验检测ScpB蛋白抗血清的保护性功能。结果成功表达并纯化了ScpB蛋白；质谱分析和蛋白质库比较证实其为B族链球菌表面蛋白C5a肽酶的可能性分值为175；ELISA显示ScpB蛋白皮下30μg及鼻内不同剂量（5μg、10μg、30μg）免疫后均可诱发小鼠产生高水平特异性IgG抗体（P〈0．01），30μg剂量组IgG大于5μg及10μg剂量组（P〈0．05）；鼻内不同剂量（5μg、10μg、30μg）免疫后均可诱发小鼠产生高水平的黏膜特异性IgA抗体，与对照组相比差异有统计学意义（P〈0．01）；黏膜免疫组间无差异；30μg ScpB蛋白皮下免疫后黏膜特异性IgA抗体与对照组相比差异无统计学意义（P〉0．05）。ScpB抗血清对细菌具有调理吞噬作用，吞噬指数为12．3，与对照组相比差异有统计学意义（P〈0．05）。结论B族链球菌表面蛋白C5a肽酶鼻内途径免疫小鼠后可在血清、肺及阴道、直肠等免疫近端和远端黏膜中产生相应高水平IgG及IgA抗体，上述ScpB抗体具有促进吞噬细胞吞噬B族链球菌的功能。

Objective To study the system of streptococcal C5a peptidase （ScpB） and the specific antibody levels in serum, lung, vagina and recta after subcutaneous and intranasal immunization with dif- ferent doses of CSa peptide. Methods Recombinant protein CSa peptide was expressed in E. coli strain BI21 and purified by affinity chromatography. The expressed product was identified by SDS-PAGE and peptide mass fingerprinting （PMF）. BALB/c mice were subcutaneously and intranasally injected with different doses of ScpB. Antibody titer was tested by ELISA. Opsonophagocytosis assay was used to test the function of antibody. Results ScpB protein was successfully expressed and purified. The probability based mouse score of ScpB was 175 by PMF analysis. ELISA data showed that both subcutaneous and intranasal immuni- zation could elicit significantly higher levels of IgG in immunized mice serum than that of control group （P〈0.01） , 30μg group was better than 5 μg and 10 μg group. Intranasal immunization could elicit higher lev- els of IgA in lung, vagina and rectum （P〈0.001） while system immunization could not. Opsonophagncytosis tests indicated that anti-serum of ScpB had opsonophagocytic activity than that of control （P〈0.05）. Conclusion The results demonstrated that intranasal immunization with ScpB could induce significantly higher levels of IgG and IgA, and its anti-serum had better opsonic activity.