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

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

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1×109CFU/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。

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 × 10^9 CFU / m l 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 purifi-cation of anti P. intermedia . SDS-PAGE gel electrophoresis, Western blot and E L ISA were used to detect mo-lecular weight , specificity and changes in the immune titer of anti P. mterjnedia-IgY. 30 female S D rats, 15 male SD rats, female ？ male = 2＊1 caged for 12 hours. 24 pregnant mice were randomly divided into 4 groups： 0. 1 2 % chlorhexidine incubation group, anti P. i nt er medi a incubation group, physiological saline incubation group and blank control group, to observe the preventive effects of anti P. intermediaon experimental pregnancy gingivi-tis. 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 recognition and specificity combined with P. intermedia bacterial cleavage fragment. Observation of 0. 12% chlorhexidine incubation group and anti P. intermedia-I^Y 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 ob-served 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 can be harvested,and antiP. intermedia-IgY can reduce the experimental pregnancy gingivitis inflammatory state of rats, which has a certain preventive effect onexperimental pregnancy gingivitis of rats.