本文選題：卵黃抗體　切入點(diǎn)：Solobacterium　出處：《大連理工大學(xué)》2011年碩士論文

【摘要】：口臭是影響人們?nèi)粘Ｉ缃患吧畹囊环N常見疾病。大量研究證實(shí)Solobacterium moorei與口臭有著緊密聯(lián)系,并確定S. moorei為口臭致病菌之一。本研究以S. moorei全菌為免疫原免疫來航蛋雞。分離卵黃,經(jīng)水稀釋,硫酸銨鹽析、硫酸鈉鹽析、超濾等分離純化IgY。分析SDS-PAGE電泳結(jié)果表明,IgY純度為87.3%。ELISA檢測(cè)IgY效價(jià)結(jié)果可見,三次免疫后第十天的抗體效價(jià)達(dá)到最高,為1：10000,且可持續(xù)2周�？筍. moorei特異性IgY與其他主要口臭致病菌牙齦卟啉單胞菌、具核梭桿菌、中間普氏菌有很弱的交叉反應(yīng),說明抗S. moorei IgY具有很強(qiáng)的特異性。 使用含抗S.moorei特異性IgY的GAM液體培養(yǎng)基培養(yǎng)S. moorei,每隔12h于600nm處檢測(cè)OD值,分析IgY對(duì)S. moorei生長(zhǎng)的影響。結(jié)果顯示,20 mg/ml及40 mg/ml的IgY均能顯著抑制S. moorei的生長(zhǎng)(P0.05),且40 mg/ml的IgY作用效果更強(qiáng)。通過菌體結(jié)晶紫染色的方法檢測(cè)IgY對(duì)S. moorei生物被膜形成的抑制作用。結(jié)果顯示,20 mg/ml及40 mg/ml的IgY均能顯著抑制S. moorei生物被膜的形成(P0.05),且有一定劑量依賴性。免疫熒光與免疫電鏡結(jié)果表明,IgY是通過與菌體的特異性結(jié)合發(fā)揮抑制作用,但并不改變菌體形態(tài)。 每隔兩天給小鼠口腔內(nèi)接種一次109CFU/ml的S. moorei 0.1ml,持續(xù)一周。在攻毒前一天及攻毒完成次日取小鼠口腔樣本,涂布于JCM 14號(hào)培養(yǎng)基及OHOC培養(yǎng)基,以測(cè)定小鼠口腔內(nèi)厭氧菌總數(shù)及S. moorei的數(shù)量變化,鑒定S. moorei是否成功定植。攻毒完成后,每隔5天口腔灌服0.1 m1濃度為40 mg/ml的特異性IgY,持續(xù)6周后,取口腔樣本涂布于JCM 14號(hào)培養(yǎng)基及OHOC培養(yǎng)基中進(jìn)行統(tǒng)計(jì)。結(jié)果表明IgY組與陰性對(duì)照組相比,小鼠口腔內(nèi)厭氧菌及S. moorei的數(shù)量顯著降低(P0.05)。檢測(cè)小鼠血液中的超氧化物歧化酶(SOD)和谷胱甘肽過氧化物酶(GSH-PX)活性以及溶菌酶和丙二醛(MDA)含量結(jié)果顯示,飼喂IgY小鼠的超氧化物歧化酶、谷胱甘肽過氧化物酶活性和溶菌酶含量顯著提高而丙二醛含量明顯降低,說明IgY能提高機(jī)體抗氧化能力及免疫力。 本研究制備了抗S. moorei特異性卵黃抗體,并證實(shí)IgY在體外和體內(nèi)都能對(duì)S. moorei有顯著的抑制作用,為防治S. moorei感染引起的口臭提供了新的途徑。
[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.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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