›› 2008, Vol. 51 ›› Issue (11): 1144-1150.

• 研究论文 • 上一篇    下一篇

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

吴玛莉,朱家颖,胡萃,叶恭银   

  • 出版日期:2008-11-20 发布日期:2008-11-20
  • 通讯作者: 叶恭银

Comparison of different methods for extraction and isolation of venom proteins from Pteromalus puparum (Hymenoptera: Pteromalidae)

WU Ma-Li   

  • Online:2008-11-20 Published:2008-11-20

摘要:

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明:等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45~116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24~116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。

关键词: 蝶蛹金小蜂, 菜粉蝶, 毒液, 分离, 血细胞延展, 血细胞包囊

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