抗牙龈卟啉单胞菌IgY的制备和体外研究

发布时间：2018-04-04 19:03

  本文选题：牙龈卟啉单胞菌　切入点：卵黄免疫球蛋白　出处：《安徽医科大学》2010年硕士论文

【摘要】： 目的将牙龈卟啉单胞菌作为抗原免疫蛋鸡,从鸡蛋中制备、纯化抗牙龈卟啉单胞菌卵黄免疫球蛋白(IgY),对其进行特异性鉴定,研究抗体滴度随免疫时间、温度、酸碱的变化,评价IgY的理化性质,并体外研究IgY对牙龈卟啉单胞菌的抑制作用。 方法厌氧培养牙龈卟啉单胞菌国际标准菌株ATCC 33277,收集细菌,超声震碎至菌体完全破碎,离心,收集上清液,即为全菌可溶性蛋白抗原。罗曼母鸡隔离饲养至5月龄用于抗体的制备。以浓度为1mg/ml牙龈卟啉单胞菌全菌可溶性蛋白抗原1ml与等量福氏完全佐剂吹打混匀成油包水状注射剂,经皮下和肌肉多点注射免疫母鸡,共注射免疫4次,每次免疫间隔时间为10天。初次免疫后即开始收集鸡蛋,标记后4℃保存。提取、纯化免疫后不同时间的抗牙龈卟啉单胞菌IgY,二喹啉甲酸总蛋白定量法(BCA法)测定蛋白浓度,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析鉴定,间接ELISA进行IgY抗体效价变化和理化性质的检测,液体培养抑菌法检测特异性IgY对牙龈卟啉单胞菌的体外抑制作用。 结果通过免疫诱导出的浓度为2.05mg/ml的特异性IgY,经SDS-PAGE检测,分子量与理论值一致;间接ELISA显示初次免疫后5天左右出现抗牙龈卟啉单胞菌IgY,免疫后50--55天到达高峰;抗体效价达到1:100 000以上;一个鸡蛋可以生产超过10mg的IgY,纯度高达95%;IgY对牙龈卟啉单胞菌具有特异性的抑菌作用,在浓度1.0 mg/ml时到达最大,抑制细菌生长率为47.21%。 结论牙龈卟啉单胞菌全菌蛋白免疫产蛋母鸡可使鸡蛋产生高效价、高浓度的特异性IgY,该抗体稳定性好,能明显抑制牙龈卟啉单胞菌的生长。
[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，

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