金黄色葡萄球菌凝集因子B的原核表达及其抗血清调理吞噬活性  

Prokaryotic expression of Staphylococcus aureus Clumping factor B and evaluation of the antiserum-mediated opsonic activity

作　　者：张丽丽[1] 曹珊珊 张屹峰 宣春玲 吴有强 胡松华[1] 

机构地区：[1]浙江大学动物科学学院,浙江杭州310012 [2]浙江海隆生物科技有限公司,浙江绍兴312000

出　　处：《生物工程学报》2015年第11期1623-1631,共9页Chinese Journal of Biotechnology

摘　　要：金黄色葡萄球菌是导致医院院内感染的主要病原。由于金黄色葡萄球菌极易产生抗药性,因此疫苗免疫是预防该细菌感染的主要手段。作为一个粘附分子,凝集因子B(ClfB)的作用是使金黄色葡萄球菌能够在宿主黏膜定植,是预防该菌感染的一个重要的靶分子。本研究成功地在大肠杆菌中表达了可溶的ClfB N1-N3结构域蛋白(Truncated-ClfB),并且利用亲和层析、离子交换层析和凝胶过滤技术对其进行了纯化。用纯化后的Truncated-ClfB免疫新西兰大白兔,收集三免后的血清检测其抗体水平并且利用流式细胞术检测抗血清的调理吞噬活性。检测结果表明,三免后的兔源Truncated-ClfB抗血清抗体效价高达1:640 000;与免疫前兔源血清相比,兔源Truncated-ClfB抗血清能够显著增加多形核白细胞(Polymorphonuclear leukocytes,PMN)对金黄色葡萄球菌的吞噬效率(P<0.01)。结果表明Truncated-Clf B有希望作为金黄色葡萄球菌疫苗的候选抗原。Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B（ClfB） is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein（Truncated-ClfB） cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-Clf B. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum Ig G titer reached 1：640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-Clf B immunized rabbits than those from no immunized group（P〈0.01）. Therefore, the results indicated that Truncated-Clf B could be a promising vaccine candidate against S. aureus infection.

关 键 词：蛋白 纯化 多形核白细胞 免疫 流式细胞术 

分 类 号：R378[医药卫生—病原生物学] Q78[医药卫生—基础医学]