基于O特异链亲和纯化特异性IgY的研究
发布时间：2018-09-11 15:05
【摘要】：卵黄抗体(immunoglobulin Y, IgY)与哺乳动物抗体IgG (immunoglobulin G)相比具有产量高、稳定性强、交叉反应低等优势,在疾病的免疫检测和防治方面具有良好的应用前景,但是在总IgY中难以分离纯化特异性IgY。因此,本论文将大肠杆菌O157作为抗原免疫蛋鸡获得特异性IgY之后,以大肠杆菌O157的O特异侧链作为亲和配基,分离纯化特异性IgY,并对其进行了表征。 本研究采用大肠杆菌O157全菌灭活疫苗免疫蛋鸡制备特异性IgY。经间接ELISA法测得,免疫后的第7周抗体效价达到最高水平,为1：128000,第11周开始抗体效价出现明显的下降趋势。收集第7至第11周的鸡蛋,用水稀释法和硫酸铵盐析法提取总IgY,基于SDS-PAGE用Lab-Image软件分析可知IgY纯度为48.3%。 O特异侧链是革兰氏阴性细菌细胞壁中脂多糖的组成成分,是免疫学分类基础。O特异侧链在同一种属内部和不同菌种之间都存在较大的差异,因此基于该特点,本研究以脂多糖作为亲和配基对总IgY中的特异性IgY进行分离纯化。实验采用超声破碎、热酚水法和超速离心技术,提取大肠杆菌O157的脂多糖总量为菌体干重的0.3%,纯度为99%。 将纯化得到的脂多糖与Epoxy活化琼脂糖凝胶FF进行偶联、装柱,用于分离纯化特异性IgY。依据凝胶的活化密度以及参考文献,使7g琼脂糖凝胶与10mg脂多糖在NaOH-KCl溶液中偶联20小时可得到较理想的偶联效果,载体偶联率为0.66 mg LPS/mL载体。将粗分离的IgY饱和上样到亲和层析柱中,然后洗脱并收集,检测蛋白含量,结果表明亲和层析柱的载量为0.75mg/mL介质。通过优化确定洗脱特异性IgY的缓冲液为Tris-HCl,浓度为0.1M、pH为8.9。间接ELISA和竞争ELISA法检测纯化后特异性IgY的抗体效价,结果显示亲和层析分离纯化得到的特异性IgY的抗体效价比纯化前提高了3倍,抗体纯度提高了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.
【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
[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.
【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
【参考文献】
相关期刊论文 前10条
1 王海龄;钟峻;肖小璞;李和;于R，
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