本文選題：中間普氏菌　切入點：卵黃抗體　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的制備并純化抗中間普氏菌卵黃抗體(抗P.intermedia-Ig Y),觀察其對大鼠實驗性妊娠期齦炎的預(yù)防作用。方法厭氧培養(yǎng)P.intermedia菌,2%甲醛滅活,以終濃度為1 x 109 CFU/m L的P.intermedia菌,1 m L(0.5 m L菌液+0.5 m L佐劑)/次,免疫蛋雞4次。用卵黃分離器去除蛋清,無菌注射器刺破卵黃膜,收集卵黃液,兩步硫銨沉淀法純化抗P.intermedia-Ig Y。SDS-PAGE凝膠電泳、Western blot、ELISA對其分子量及特異性鑒定,并檢測免疫后效價變化。將30只雌性SD大鼠(8-10周齡),15只雄性SD大鼠(8-10周齡),雌:雄=2:1合籠獲得24只孕鼠,隨機分為4組:0.12%氯己定孵育組(A),抗P.intermedia-Ig Y孵育組(B),生理鹽水孵育組(C),空白組(D)。在造模期間,按分組情況接種經(jīng)不同處理液孵育后的P.intermedia,1次/天,總共7次。通過檢測各組之間牙齦指數(shù)(GI)、牙菌斑中P.intermedia含量差異及牙齦組織病理學(xué)檢查,觀察抗P.intermedia-Ig Y對實驗性妊娠期齦炎的抑制作用。結(jié)果經(jīng)4次免疫后抗體最高效價為1:512,000,維持45 d。1 g卵黃經(jīng)兩步硫銨沉淀后得Ig Y約18 mg。SDS-PAGE凝膠電泳顯示純化后Ig Y純度達64.5%。Western blot顯示抗P.intermedia-Ig Y識別并特異性結(jié)合P.intermedia菌裂解片段。動物實驗觀察見0.12%氯己定孵育組及抗P.intermedia-Ig Y孵育組牙齦指數(shù)(gingival index,GI)顯著低于生理鹽水孵育組(P0.01),而兩者之間GI無統(tǒng)計學(xué)差異(P0.05)。牙齦組織病理學(xué)觀察顯示抗P.intermedia-Ig Y孵育組牙齦的炎癥狀態(tài)輕于生理鹽水孵育組。結(jié)論經(jīng)4次免疫后可得到持續(xù)高效價的抗P.intermedia-Ig Y,且抗P.intermedia-Ig Y能夠減輕大鼠實驗性妊娠期齦炎的炎癥狀態(tài),對大鼠實驗性妊娠期齦炎有一定的預(yù)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.4;R714.2

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