本文選題：牙齦卟啉單胞菌　切入點(diǎn)：卵黃免疫球蛋白　出處：《安徽醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的將牙齦卟啉單胞菌作為抗原免疫蛋雞,從雞蛋中制備、純化抗牙齦卟啉單胞菌卵黃免疫球蛋白(IgY),對(duì)其進(jìn)行特異性鑒定,研究抗體滴度隨免疫時(shí)間、溫度、酸堿的變化,評(píng)價(jià)IgY的理化性質(zhì),并體外研究IgY對(duì)牙齦卟啉單胞菌的抑制作用。 方法厭氧培養(yǎng)牙齦卟啉單胞菌國(guó)際標(biāo)準(zhǔn)菌株ATCC 33277,收集細(xì)菌,超聲震碎至菌體完全破碎,離心,收集上清液,即為全菌可溶性蛋白抗原。羅曼母雞隔離飼養(yǎng)至5月齡用于抗體的制備。以濃度為1mg/ml牙齦卟啉單胞菌全菌可溶性蛋白抗原1ml與等量福氏完全佐劑吹打混勻成油包水狀注射劑,經(jīng)皮下和肌肉多點(diǎn)注射免疫母雞,共注射免疫4次,每次免疫間隔時(shí)間為10天。初次免疫后即開(kāi)始收集雞蛋,標(biāo)記后4℃保存。提取、純化免疫后不同時(shí)間的抗牙齦卟啉單胞菌IgY,二喹啉甲酸總蛋白定量法(BCA法)測(cè)定蛋白濃度,十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)分析鑒定,間接ELISA進(jìn)行IgY抗體效價(jià)變化和理化性質(zhì)的檢測(cè),液體培養(yǎng)抑菌法檢測(cè)特異性IgY對(duì)牙齦卟啉單胞菌的體外抑制作用。 結(jié)果通過(guò)免疫誘導(dǎo)出的濃度為2.05mg/ml的特異性IgY,經(jīng)SDS-PAGE檢測(cè),分子量與理論值一致;間接ELISA顯示初次免疫后5天左右出現(xiàn)抗牙齦卟啉單胞菌IgY,免疫后50--55天到達(dá)高峰;抗體效價(jià)達(dá)到1:100 000以上;一個(gè)雞蛋可以生產(chǎn)超過(guò)10mg的IgY,純度高達(dá)95%;IgY對(duì)牙齦卟啉單胞菌具有特異性的抑菌作用,在濃度1.0 mg/ml時(shí)到達(dá)最大,抑制細(xì)菌生長(zhǎng)率為47.21%。 結(jié)論牙齦卟啉單胞菌全菌蛋白免疫產(chǎn)蛋母雞可使雞蛋產(chǎn)生高效價(jià)、高濃度的特異性IgY,該抗體穩(wěn)定性好,能明顯抑制牙齦卟啉單胞菌的生長(zhǎng)。
[Abstract]:Objective to prepare and purify immunoglobulin yolk immunoglobulin (IGY) against Porphyromonas gingivalis from eggs of layers immunized with porphyromonas gingivalis as antigen, and to study the changes of antibody titer with immune time, temperature, acid and base.To evaluate the physical and chemical properties of IgY and to study the inhibitory effect of IgY on Porphyromonas gingivalis in vitro.Methods ATCC 33277, an international standard strain of Porphyromonas gingivalis, was cultured in anaerobic culture. The bacteria were collected, the bacteria were crushed by ultrasound, centrifuged, supernatant was collected, which was the soluble protein antigen of the whole bacteria.Roman hens were kept in isolation until 5 months old for antibody preparation.The soluble protein antigen (1ml) of Porphyromonas gingivalis (1mg/ml) was mixed with an equal amount of Freund's complete adjuvant to form a water-in-oil injection. The hens were immunized by subcutaneous and multi-point injection, and the hens were immunized for 4 times.The interval between immunizations was 10 days.Eggs were collected and stored at 4 鈩，

本文編號(hào)：1711271