Egg Yolk Immunoglobulin Affinity Ligand To Separation Of The Study Of Snake Venom Gln49-pla <sub> 2 </ Sub>

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%.