Purification Of Recombinant Human Zona-pellucida-3(rhZP3) And Application Of RhZP3 In Detection For Antibodies Against Zona Pellucida

As zona pellucida (ZP) plays a vital role in sperm-egg recognition in the process of fertilization, it is considered to be an ideal target for immunocontraceptive vaccine. The recombinant human zona pellucida-3(rhZP3) expression vector has been constructed and expressed in Pichia Pastoris. In the present study, the main objective is exploring appropriate pH flow-rate chelated metal ion and elution buffer for the purification of rhZP3 by monitoring the yield and purity of products, and obtaining pure rhZP3 which will be used as the antigen to detect the antibodies against zona pellucida and observe immunocontraceptive potential.The pre-treated culture supernatant which the impurity in culture have been removed and the proteins have been concentrated by ultrafiltration were added on the column for immobilized metal ion affinity chromatography (IMAC) with different conditions, such as pH flow-rate immobilized metal ion. The rhZP3 were eluted with imidazole. Then the purity and yield of the rhZP3 were determined by SDS-PAGE and Western-Blotting. The purified rhZP3 was used as coating antigen in ELISA to detect the antibodies against zona pellucida in sera of 90 sterility women, and then comparing results with those of ELISA kits made from natural porcine ZP3.The results showed that as pH increase, the yield of rhZP3 raised while the purity declined; as flow-rate reduction, the yield decreased but the purity improved; the Ni²⁺-IDA column was more successful in purification of rhZP3 compared with Cu²⁺-IDA column. The yield could be increased through repurify of flow-through peak and the purity could be improved by washed with NH₄Cl before the elution with imidazole. The recovery and the purity of rhZP3 could reach 85% and 90% respectively by the optimized methods. The results of antibodies detection by ELISA using rhZP3 were consistent with those of ELISA kits from natural porcine ZP3.These results imply that the optimized conditions for purification of rhZP3 are loading the concentrated proteins on Ni²⁺-chelated column in pH7.5 at 2ml/min, washing the column with 0.5mol/L NH₄Cl at first to get rid of impurities and then eluting purified rhZP3 with 0.1mol/L imidazole. rhZP3 is as effective as natural porcine ZP3 in detecting the antibodies against zona pellucida by ELISA.