【摘要】：卵黃抗體(immunoglobulin Y, IgY)與哺乳動物抗體IgG (immunoglobulin G)相比具有產(chǎn)量高、穩(wěn)定性強、交叉反應(yīng)低等優(yōu)勢,在疾病的免疫檢測和防治方面具有良好的應(yīng)用前景,但是在總IgY中難以分離純化特異性IgY。因此,本論文將大腸桿菌O157作為抗原免疫蛋雞獲得特異性IgY之后,以大腸桿菌O157的O特異側(cè)鏈作為親和配基,分離純化特異性IgY,并對其進行了表征。 本研究采用大腸桿菌O157全菌滅活疫苗免疫蛋雞制備特異性IgY。經(jīng)間接ELISA法測得,免疫后的第7周抗體效價達到最高水平,為1：128000,第11周開始抗體效價出現(xiàn)明顯的下降趨勢。收集第7至第11周的雞蛋,用水稀釋法和硫酸銨鹽析法提取總IgY,基于SDS-PAGE用Lab-Image軟件分析可知IgY純度為48.3%。 O特異側(cè)鏈是革蘭氏陰性細菌細胞壁中脂多糖的組成成分,是免疫學(xué)分類基礎(chǔ)。O特異側(cè)鏈在同一種屬內(nèi)部和不同菌種之間都存在較大的差異,因此基于該特點,本研究以脂多糖作為親和配基對總IgY中的特異性IgY進行分離純化。實驗采用超聲破碎、熱酚水法和超速離心技術(shù),提取大腸桿菌O157的脂多糖總量為菌體干重的0.3%,純度為99%。 將純化得到的脂多糖與Epoxy活化瓊脂糖凝膠FF進行偶聯(lián)、裝柱,用于分離純化特異性IgY。依據(jù)凝膠的活化密度以及參考文獻,使7g瓊脂糖凝膠與10mg脂多糖在NaOH-KCl溶液中偶聯(lián)20小時可得到較理想的偶聯(lián)效果,載體偶聯(lián)率為0.66 mg LPS/mL載體。將粗分離的IgY飽和上樣到親和層析柱中,然后洗脫并收集,檢測蛋白含量,結(jié)果表明親和層析柱的載量為0.75mg/mL介質(zhì)。通過優(yōu)化確定洗脫特異性IgY的緩沖液為Tris-HCl,濃度為0.1M、pH為8.9。間接ELISA和競爭ELISA法檢測純化后特異性IgY的抗體效價,結(jié)果顯示親和層析分離純化得到的特異性IgY的抗體效價比純化前提高了3倍,抗體純度提高了50倍。
[Abstract]:Compared with mammalian antibody IgG (immunoglobulin G), yolk antibody (immunoglobulin Y, IgY) has the advantages of high yield, strong stability and low cross-reaction. However, it is difficult to isolate and purify specific IgY. from total IgY. Therefore, the specific IgY of Escherichia coli O157 was purified and characterized by using the O specific side chain of E. coli O157 as the affinity ligand after immunizing laying hens with Escherichia coli O157 as antigen. Preparation of specific IgY. from laying hens immunized with Escherichia coli O157 inactivated vaccine Indirect ELISA assay showed that the titer of antibody reached the highest level at the 7th week after immunization, which was 1: 128000, and the titer of antibody decreased obviously at the 11th week. Collecting eggs from week 7 to 11, extracting total IgY, by water dilution method and ammonium sulfate salting-out method. Based on SDS-PAGE analysis by Lab-Image software, the purity of IgY was 48.3%. O specific side chain was a component of lipopolysaccharide in the cell wall of Gram-negative bacteria. It is the basis of immunological taxonomy. The specific side chain of .O is different within the same genus and among different strains. Therefore, lipopolysaccharide was used as the affinity ligand to isolate and purify the specific IgY from the total IgY. The total lipopolysaccharide of Escherichia coli O157 was extracted by ultrasonic crushing, thermophenol water method and ultracentrifugation. The total lipopolysaccharide of E. coli O157 was 0.33% of the dry weight of bacteria, and the purity was 9910%. The purified lipopolysaccharide was coupled with Epoxy activated agarose gel FF and packed with column for the separation and purification of specific IgY.. According to the activation density of the gel and the reference, the coupling effect of 7 g agarose gel with 10mg lipopolysaccharide in NaOH-KCl solution for 20 hours was satisfactory, and the coupling rate of the carrier was 0.66 mg LPS/mL. The saturated sample of crude IgY was added to the affinity chromatography column, then eluted and collected, and the protein content was detected. The results showed that the load of the affinity chromatography column was 0.75mg/mL medium. The buffer solution for elution of specific IgY was determined by optimization. The concentration of Tris-HCl, was 0.1 MN and pH was 8.9. Indirect ELISA and competitive ELISA were used to detect the antibody titers of the purified specific IgY. The results showed that the antibody titers of the specific IgY purified by affinity chromatography were increased by 3 times and the purity of the antibodies were 50 times higher than those before purification.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【參考文獻】

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本文編號：2237042