Abstract: For developing a suitable isolation and purification method of Pteromalus puparum venom proteins with the activity to inhibit host hemocyte spreading and encapsulation, seven methods, namely, isoelectric precipitation, ethanol precipitation, 75% ammonium sulphate precipitation, 75% ammonium sulphate precipitation combined with heating at 40℃, 75% ammonium sulphate precipitation combined with three different centrifugal size exclusion membranes (molecular weight cut-off exclusion limit: 10, 50 and 100 kDa), were compared. The results indicated that the isoelectric precipitated fractions had the highest activity to inhibit the spreading and encapsulation of host Pieris rapae hemocytes in vitro, the ethanol precipitated fractions acted the second, while 75% ammonium sulphate precipitated fractions showed the lowest. The results of SDS-PAGE analysis demonstrated that the purity of the isoelectric precipitated fractions was the highest with only three main protein bands with apparent molecular masses ranging from 45 to 116.2 kDa, the ethanol precipitated fractions showed five main protein bands with apparent molecular masses ranging from 24 to 116.2 kDa, and the protein profiles of ammonium sulphate precipitated fractions had nearly identical protein profiles as crude venom. To compare the activity of venom fractions from three different centrifugal size exclusion membranes, the molecular weight of the fractions with higher activity might be more than 100 kDa. It is concluded that the method of isoelectric precipitation is relatively most suitable among the seven tested methods for isolation of P. puparum venom.

Key words: Pteromalus puparum, Pieris rapae, venom, isolation, hemocyte spreading, hemocyte encapsulation