抗中间普氏菌卵黄抗体的制备及其对实验性妊娠期龈炎的预防作用

目的 制备并纯化抗中间普氏菌卵黄抗体(抗P.intermedia-IgY),观察其对大鼠实验性妊娠期龈炎的预防作用.方法 厌氧培养P.intermedia菌,2%甲醛灭活,以终浓度为1×109 CFU/ml的P.intermedia菌,1 ml(0.5 ml菌液+0.5 ml佐剂)/次,免疫蛋鸡4次.用卵黄分离器去除蛋清,无菌注射器刺破卵黄膜,收集卵黄液,两步硫铵沉淀法纯化抗P.intermedia-IgY.SDS-PAGE凝胶电泳、Western blot、ELISA对其分子量及特异性鉴定,并检测免疫后效价变化.将30只雌性SD大鼠,15只雄性SD大鼠,雌 ∶雄=2 ∶1,合笼获得24只孕鼠,随机分为4组:0.12%氯己定孵育组,抗P.intermedia-IgY孵育组,生理盐水孵育组,空白对照组,观察抗P.intermedia-IgY对实验性妊娠期龈炎的抑制作用.结果 经4次免疫后抗体最高效价为1 ∶512 000,维持45 d.1 g卵黄经两步硫铵沉淀后得IgY约18 mg.SDS-PAGE凝胶电泳显示纯化后IgY纯度达64.5%.Western blot显示抗P.intermedia-IgY识别并特异性结合P.intermedia菌裂解片段.动物实验观察见0.12%氯己定孵育组及抗P.intermedia-IgY孵育组牙龈指数显著低于生理盐水孵育组(P<0.01),而两者之间牙龈指数差异无统计学意义.牙龈组织病理学观察显示抗P.intermedia-IgY孵育组牙龈的炎症状态轻于生理盐水孵育组.结论 经4次免疫后可得到持续高效价的抗P.intermedia-IgY,且抗P.intermedia-IgY能够减轻大鼠实验性妊娠期龈炎的炎症状态,对大鼠实验性妊娠期龈炎有一定的预防作用.

Preparation of egg york antibody aganist Prevotella intermediaand its preventive effect on expeimental pregnancy gingivitis

Objective To prepare and purify anti Prevotella intermedia yolk antibody (anti P.intermedia-IgY), and observe its preventive effect on rat experimental pregnancy gingivitis.Methods P.intermedia was cultured anaerobically, inactivated with 2% formaldehyde,with final concentration of 1×109 CFU/ml bacteria, 1 ml(0.5 ml liquid+0.5 ml adjuvant)/times, 4 times the immune layer.Removed the egg white with yolk separator, sterile syringe punctured the vitelline membrane, collecting the yolk, two step ammonium sulfate precipitation and purification of anti P.intermedia-IgY.SDS-PAGE gel electrophoresis, Western blot and ELISA were used to detect molecular weight , specificity and changes in the immune titer of anti P.intermedia-IgY.30 female SD rats, 15 male SD rats, female ∶male=2 ∶1 caged for 12 hours.24 pregnant mice were randomly divided into 4 groups: 0.12% chlorhexidine incubation group, anti P.intermedia-IgY incubation group, physiological saline incubation group and blank control group, to observe the preventive effects of anti P.intermedia-IgY on experimental pregnancy gingivitis.Results After 4 immunization, the highest titer of antibody was 1 ∶512 000, which was maintained at 45 d.1 g yolk by two step ammonium sulfate precipitation was IgY about 18 mg.SDS-PAGE gel electrophoresis showed that the purity of purified IgY was 64.5%.Western blot showed anti P.intermedia-IgY recognition and specificity combined with P.intermedia bacterial cleavage fragment.Observation of 0.12% chlorhexidine incubation group and anti P.intermedia-IgY incubation group gingival index were significantly lower than that of the saline incubation group (P<0.01), but there was no difference between the two gingival index.Gingival tissue pathology was observed to show that the inflammatory state of the anti P.intermedia-IgY group was less than that of the saline group.Conclusion After 4 times of immunization, sustaining high titer anti P.intermedia-IgY can be harvested,and anti P.intermedia-IgY can reduce the experimental pregnancy gingivitis inflammatory state of rats, which has a certain preventive effect onexperimental pregnancy gingivitis of rats.