[Acquisition and the immunogenicity of the outer membrane FomA protein of Fusobacterium nucleatum].

Objective: To express and purify outer membrane protein FomA of Fusobacterium nucleatum (Fn) through gene recombination technique with Escherichia coli (Ec) expression system, and to detect the immunogenicity and the immune effects of the recombinant protein on gingival tissues. Methods: The gene recombination technique and Ec expression system were used to express and purified the FomA protein. Totally 20 C57 mice were immuned with the protein or the phosphate buffer solution (PBS) buffer by subcutaneous injection (each 10 mice), and the specific FomA antibody was detection in mice serum. The immunogenicity of FomA protein was assessed by comparing the differences between groups. Furthermore, the model of mice gum abscess was constructed with Fn or Fn and Porphyromonas gingivalis (Pg) mixed suspension used the above mice. The score of the gingival abscess was recorded and the interleukin (IL)-1β in gum tissue and mice serum was determined by enzyme-linked immunosorbent assay method, and the differences of the indexes between groups were compared to evaluate the effect of the FomA protein immunization. Results: Totally 1.0-1.5 g FomA protein were successfully obtained and the protein purity was over 90%. The FomA specific antibody was detected in the serum of mice by subcutaneous injection of the protein, and the antibody titer reached the highest level in 2 weeks after secondary immunization. The model of submaxillary gingival abscess was successfully constructed. In the Fn model, the score of the FomA protein immune group was (1.82±0.35), and the PBS control group was (2.62±0.71), with statistically significant difference between the two groups (P=0.049). In the Fn+Pg mixture model, the score of gingival abscess in the FomA immune group (2.31±0.55) was lower than that in PBS group (3.63±0.45), and the difference was statistically significant (P=0.003). Both in Fn and Fn+Pg injection group, the concentration of IL-1β in the serum of FomA immune mice and gingival tissues was lower than that of PBS control mice (P<0.001). Conclusions: The recombinant FomA protein can be acquired by Ec expression system, and it can produce a certain level antibodies in the mice serum. The way of mice subcutaneously injected with the recombinant FomA protein can reduce the severity of periodontal infections caused by Fn and Pg.目的： 检测具核梭杆菌（Fusobacterium nucleatum，Fn）的主要外膜孔道FomA蛋白的免疫原性、免疫水平以及对牙龈组织的免疫效果，为研究以FomA蛋白为靶点预防及治疗牙周感染的可行性提供理论依据。 方法： 应用基因重组技术，使用大肠杆菌表达系统表达并获取纯化的FomA蛋白，将50 μg该蛋白或50 ml磷酸盐缓冲液（phosphate buffer solution，PBS）通过皮下注射方式免疫C57小鼠（随机数字法分组，每组各10只），在小鼠血清中检测FomA特异性抗体表达情况，比较组间差异，评估FomA蛋白的免疫原性。再将上述小鼠分成FomA Fn组、FomA Fn+牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）组、PBS Fn组、PBS Fn+Pg组，分别使用Fn菌悬液或Fn与Pg混合菌悬液制备牙周脓肿模型（PBS为相应FomA组的对照组），对小鼠牙龈脓肿评分，酶联免疫吸附测定法测定小鼠血清和牙龈组织中白介素（interleukin，IL）1β的浓度，比较上述指标的组间差异，评价FomA蛋白的免疫效果。 结果： 通过基因重组技术成功获取1.0~1.5 g FomA蛋白，将该蛋白皮下注射免疫小鼠后，小鼠血清中均可检测到FomA特异性抗体，该抗体效价在二次免疫后2周达峰值。利用牙周致病菌Fn及Fn+Pg成功构建小鼠下颌牙龈脓肿模型，FomA Fn组小鼠牙龈脓肿评分[（1.82±0.35）分]显著低于PBS Fn组[（2.62±0.71）分]（P=0.049）；FomA Fn+Pg组小鼠牙龈脓肿评分[（2.31±0.55）分]亦显著低于PBS Fn+Pg组[（3.63±0.45）分]（P=0.003）；FomA Fn+Pg组与FomA Fn组小鼠血清及牙龈组织中IL-1β浓度均分别显著低于PBS对照组（P<0.001）。 结论： 使用大肠杆菌表达系统能成功表达并获得一定量的Fn外膜FomA蛋白，该重组蛋白具有一定的免疫原性，通过对小鼠皮下注射的方式可使小鼠血清中产生一定水平的抗体，有助于机体降低牙周致病菌Fn及Pg引起的牙周感染程度。.