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

发布时间：2018-03-19 08:58

本文选题：中间普氏菌　切入点：卵黄抗体　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的制备并纯化抗中间普氏菌卵黄抗体(抗P.intermedia-Ig Y),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 x 109 CFU/m L的P.intermedia菌,1 m L(0.5 m L菌液+0.5 m L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-Ig Y。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(8-10周龄),15只雄性SD大鼠(8-10周龄),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(A),抗P.intermedia-Ig Y孵育组(B),生理盐水孵育组(C),空白组(D)。在造模期间,按分组情况接种经不同处理液孵育后的P.intermedia,1次/天,总共7次。通过检测各组之间牙龈指数(GI)、牙菌斑中P.intermedia含量差异及牙龈组织病理学检查,观察抗P.intermedia-Ig Y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Ig Y约18 mg。SDS-PAGE凝胶电泳显示纯化后Ig Y纯度达64.5%。Western blot显示抗P.intermedia-Ig Y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-Ig Y孵育组牙龈指数(gingival index,GI)显著低于生理盐水孵育组(P0.01),而两者之间GI无统计学差异(P0.05)。牙龈组织病理学观察显示抗P.intermedia-Ig Y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-Ig Y,且抗P.intermedia-Ig Y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。
[Abstract]:Objective to prepare and purify anti-P.intermedia-Ig yolk antibody and observe its preventive effect on experimental gestational gingivitis in rats. Methods 2% formaldehyde was inactivated by anaerobic culture of P.intermedia. The egg white was removed with egg yolk separator and the egg yolk membrane was punctured with sterile syringe to collect egg yolk liquid with 1 脳 109 CFU/m / L P. intermedia 1 mL / L 0.5 mL adjuvant / time and 4 times immunized laying hens with the final concentration of 1 脳 109 CFU/m / L P. intermedia strain 1 mL / L and 0.5 mL / L respectively, and the egg yolk solution was collected by the sterile syringe. The molecular weight and specificity of anti-P.intermedia-Ig Y.SDS-PAGE gel electrophoresis were purified by two-step ammonium sulfate precipitation method, and the titer changes after immunization were detected. Twenty-four pregnant rats were obtained from 30 female SD rats aged 8-10 weeks, 15 male SD rats aged 8-10 weeks, female: male 2: 1 cage. The rats were randomly divided into 4 groups: 0. 12% chlorhexidine incubating group, anti P. intermedia-Ig Y incubating group, normal saline incubating group, and blank group. During the modeling period, P.intermedia was inoculated once a day after different treatments. The gingival index (GI), the content difference of P. intermedia in dental plaque and the histopathological examination of gingival were measured. The inhibitory effect of anti-P.intermedia-Ig Y on experimental gingivitis during pregnancy was observed. Results the highest titer of antibody was 1: 5120.Results after 4 times immunization, the highest titer of antibody was 1: 5120.The purified IgG Y was obtained from 45 d 1.1g egg yolk precipitated by two-step ammonium thionium. The purified IgG Y was obtained by gel electrophoresis for about 18 mg.SDS-PAGE. The gingival index of 0.12% chlorhexidine incubated group and anti-P.intermedia-Ig Y group was significantly lower than that of normal saline incubation group (P 0.01), but there was no GI between them. The pathological observation of gingival histopathology showed that the inflammation state of gingiva in the anti-P.intermedia-Ig Y group was lighter than that in the saline group. Conclusion after 4 times of immunization, the anti-P.intermedia-Ig Y can be obtained, and the anti-P.intermedia-Ig Y can reduce the rat solid state. The inflammatory state of gestational gingivitis, It has certain preventive effect on experimental pregnancy gingivitis in rats.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4;R714.2

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