Purification of phospholipase A_2 homologue from Agkistrodon blomhoffii ussurensis snake venom using an affinity ligand from immunoglobulin yolk

摘要

A new efficient affinity ligand of immunoglobulin yolk (IgY) was made and used in the purification of phospholipase A_2 (Gln49-PLA_2) homologue from Agkistrodon blomhoffii ussurensis snake venom. Polyclonal antibodies specific against PLA_2 homologue were prepared by immunize hens with PLA_2 homologue as antigen and isolated from egg yolk by an immunoaffinity column coupled with PLA_2 homologue immobilized on Sepharose 4 fast flow. SDS-PAGE and Western-blot analysis confirmed that anti-PLA_2 antibody was electrophoretically pure and specific and its activity was increased 40 times. Then, specific immunoglobulin yolk was collected and immobilized on Sepharose 4 fast flow as an immunoglobulin ligand to purify PLA_2 homologue from A.b. ussurensis snake venom and the product displayed just one band on SDS-PAGE. The capacity of IgY immunoaffinity column was determined. The recovery of PLA_2 homologue is over 83% and the productivity is about 13%, the maximum binding capacity of the IgY ligand is 0.3 mg PLA_2 homologue/ml gel. This suggests that the immunoaffinity ligand of IgY is efficient and stable.