卵黄免疫球蛋白亲和配基分离蛇毒Gln49-PLA₂的研究

【作者】 王翔宇；

【导师】 安利佳；

【作者基本信息】 大连理工大学 ， 生物化工， 2004， 硕士

【摘要】 免疫亲和层析是一种非常有效的生物大分子纯化方法，特别是应用在纯化过程的第一步。本论文根据卵黄免疫球蛋白（IgY）产量大、易于分离等特点，将其作为免疫亲和配基分离相应抗原。 选用长白山白眉蝮蛇蛇毒中的Gln49磷脂酶A₂（Gln49-PLA₂）为抗原免疫产蛋鸡，收集鸡蛋，以天数和ELISA数值（OD值表示IgY相对活性）为坐标绘制免疫滴定曲线，结果表明三次加强免疫后可持续获得抗体，在免疫后51天相对活性达到最高（0.654），一年后抗体的相对活性维持在0.614，证明特异性抗体的含量至少在一年内基本不变。 几种PEG6000沉淀法粗分IgY的结果显示卵黄液采用PEG6000-乙醇沉淀法，得到的IgY纯度最高，特异活性强，卵磷脂去除较彻底，利于长期保存；用偶联Gln49-PLA₂的Sepharose 4 Fast Flow的亲和介质从卵黄液中一步分离得到特异性抗体，抗体活性提高39倍；以抗GIn49-PLA₂-Sepharose 4 Fast Flow的特异性IgY重复上样，收集相应组分，根据Langmuir-type吸附等温曲线，测定免疫亲和介质的最大载量qm为0.59 mg/mlgel，解离常数K_d为4.4×10^-5mg/ml；Western-blot显示抗原与抗体特异性结合。 以抗GIn49-PLA₂特异性IgY为配基偶联到溴化氰活化的Sepharose 4 Fast Flow介质上，一步分离长白山白眉蝮蛇粗毒中的Gln49-PLA₂，所得目的蛋白纯度高达95％，回收率大于83％，产率约为13％，与实验室四步分离Gln49-PLA₂相比，步骤简单，产率提高6.5倍；Anti-Gln49-PLA₂-IgY-Sepharose 4 Fast Flow亲和介质的最大载量qm为0.49mg/ml gel，解离常数K_d为1.66×10^-7mg/ml，表明以anti-Gln49-PLA2特异性IgY为配基时不需要苛刻的洗脱条件，是比较理想的生物亲和配基；配基使用60次后，性质仍保持稳定无泄漏情况发生。 以Anti-Gln49-PLA2特异性IgY作为配基纯化大肠杆菌中表达的重组Gln49-PLA2，目的蛋白纯度从4.98％提高到11.5％。更多还原

【Abstract】 Immunoaffinity chromatography (IAC) is one of the efficient purification methods on biomacromolecule, especially in the first purifying process. Studies on immunoglobulin yolk (IgY) as ligand are carried out, which are based on IgY features that it has a large amount and is easy to isolate.Hens are immunized using Gln49 phospholipase A2 (Gln49-PLA2) from Agkistrodon blomhoffii ussurensis (Changbai mountain Agkistrodon Halys) snake venom as antigen. Antibody ELISA value at the concentration of 400 ug /ml reaches a peak (0.654) in the 51st day, and has maintained almost as high as 0.614 a year later. Partial purification is conducted according to several kinds of methods of PEG6000. The purity and specific activity is higher by the method of PEG6000, and plenty of phospholipid is got rid of better, which is benefit to store for a long time. Immunoaffinity column (Sepharose 4 Fast Flow) is made with Gln49-PLA2 and used to isolate anti-Gln49-PLA2 antibody from egg yolk. The antibody activity is increased by 39 times. SDS-PAGE and Western-blot analysis confirm that antibody isolated from egg yolk is electrophoretically pure (95%) and specific. By applying various amounts of IgY specific against Gln49-PLA2 to the column, the binding capacity (qm) and dissociation constant (Kd) are 0.59 mg/ml gel and 4.4 X 10-5 mg/ml, respectively, as determined by Langmuir-type adsorption isotherms.Using specific IgY as ligand, the pure targeted protein (Gln49-PLA2) is obtained, and its purity is just one band on SDS-PAGE. The recovery of Gln49-PLA2 is higher than 83%, and the productivity is about 13%, which is as 6.5 times as Gln49-PLA2by four separating steps. The Kd and qm of the anti-Gln49-PLA2-IgY immunoaffinity column are 1.66X10-7 mg/ml and 0.49 mg/ml gel.The purity of Gln-49PLA2 expressed in E. coli is increased from 4.98% to 11.5%.更多还原