抗口臭致病菌Solobacterium moorei特异性IgY的活性研究

发布时间：2018-04-03 09:11

  本文选题：卵黄抗体　切入点：Solobacterium　出处：《大连理工大学》2011年硕士论文

【摘要】：口臭是影响人们日常社交及生活的一种常见疾病。大量研究证实Solobacterium moorei与口臭有着紧密联系,并确定S. moorei为口臭致病菌之一。本研究以S. moorei全菌为免疫原免疫来航蛋鸡。分离卵黄,经水稀释,硫酸铵盐析、硫酸钠盐析、超滤等分离纯化IgY。分析SDS-PAGE电泳结果表明,IgY纯度为87.3%。ELISA检测IgY效价结果可见,三次免疫后第十天的抗体效价达到最高,为1：10000,且可持续2周。抗S. moorei特异性IgY与其他主要口臭致病菌牙龈卟啉单胞菌、具核梭杆菌、中间普氏菌有很弱的交叉反应,说明抗S. moorei IgY具有很强的特异性。 使用含抗S.moorei特异性IgY的GAM液体培养基培养S. moorei,每隔12h于600nm处检测OD值,分析IgY对S. moorei生长的影响。结果显示,20 mg/ml及40 mg/ml的IgY均能显著抑制S. moorei的生长(P0.05),且40 mg/ml的IgY作用效果更强。通过菌体结晶紫染色的方法检测IgY对S. moorei生物被膜形成的抑制作用。结果显示,20 mg/ml及40 mg/ml的IgY均能显著抑制S. moorei生物被膜的形成(P0.05),且有一定剂量依赖性。免疫荧光与免疫电镜结果表明,IgY是通过与菌体的特异性结合发挥抑制作用,但并不改变菌体形态。 每隔两天给小鼠口腔内接种一次109CFU/ml的S. moorei 0.1ml,持续一周。在攻毒前一天及攻毒完成次日取小鼠口腔样本,涂布于JCM 14号培养基及OHOC培养基,以测定小鼠口腔内厌氧菌总数及S. moorei的数量变化,鉴定S. moorei是否成功定植。攻毒完成后,每隔5天口腔灌服0.1 m1浓度为40 mg/ml的特异性IgY,持续6周后,取口腔样本涂布于JCM 14号培养基及OHOC培养基中进行统计。结果表明IgY组与阴性对照组相比,小鼠口腔内厌氧菌及S. moorei的数量显著降低(P0.05)。检测小鼠血液中的超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性以及溶菌酶和丙二醛(MDA)含量结果显示,饲喂IgY小鼠的超氧化物歧化酶、谷胱甘肽过氧化物酶活性和溶菌酶含量显著提高而丙二醛含量明显降低,说明IgY能提高机体抗氧化能力及免疫力。 本研究制备了抗S. moorei特异性卵黄抗体,并证实IgY在体外和体内都能对S. moorei有显著的抑制作用,为防治S. moorei感染引起的口臭提供了新的途径。
[Abstract]:Halitosis is a common disease that affects people's daily life and social life.A large number of studies have confirmed that Solobacterium moorei is closely related to halitosis and identified S. moorei as one of the pathogens of halitosis.In this study, laying hens were immunized with S. moorei as immunogen.The egg yolk was separated and purified by water dilution, ammonium sulfate saltout, sodium sulfate saltout and ultrafiltration.The results of SDS-PAGE electrophoresis showed that the purity of IGY was 87.3%.ELISA to detect the titer of IgY, and the titer of the antibody reached the highest level on the 10th day after the third immunization, which was 1: 10 000, and lasted for 2 weeks.The anti-S. moorei specific IgY had a weak cross reaction with other major halitosis pathogens, Porphyromonas gingivalis, Clostridium ribosus and Propagation Intermediate, indicating that the resistance to S. moorei IgY was highly specific.S. Mooreii was cultured on GAM liquid medium containing anti S.moorei specific IgY. OD values were measured at 600nm every 12 hours to analyze the effect of IgY on the growth of S. moorei.The results showed that both IgY of 20 mg/ml and 40 mg/ml could significantly inhibit the growth of S.#en3#, and the effect of 40 mg/ml on IgY was stronger.The inhibitory effect of IgY on the formation of S. moorei biofilm was detected by means of cell crystal violet staining.The results showed that both 20 mg/ml and 40 mg/ml IgY could significantly inhibit the formation of S.#en3# biofilm in a dose-dependent manner.The results of immunofluorescence and immunoelectron microscopy showed that IGY inhibited the cell morphology by the specific binding with the bacteria, but did not change the morphology of the cell.S. moorei 0.1ml of 109CFU/ml was inoculated in mice every two days for one week.The oral samples of mice were collected one day before and the next day after the attack. The samples were coated on JCM medium 14 and OHOC medium to determine the total number of anaerobic bacteria in the oral cavity of mice and the changes of the number of S. moorei, and to determine whether S. moorei was successfully colonized.After oral administration every 5 days, the specific IgYs with a concentration of 0. 1 ml was 40 mg/ml. After 6 weeks, the oral samples were coated in JCM 14 medium and OHOC medium for statistical analysis.The results showed that the number of anaerobic bacteria and moorei in oral cavity of IgY group was significantly lower than that of negative control group (P 0.05).The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) and the contents of lysozyme and malondialdehyde (MDA) in blood of mice were determined. The results showed that the superoxide dismutase (SOD) was fed to IgY mice.Glutathione peroxidase activity and lysozyme content were significantly increased, while malondialdehyde content was significantly decreased, indicating that IgY can improve the antioxidant capacity and immunity.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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