›› 2009, Vol. 52 ›› Issue (6): 624-630.doi:

• 研究论文 • 上一篇    下一篇


阎岩, 吴建伟, 罗杰, 洪牧, 国果, 付萍   

  • 出版日期:2009-06-20 发布日期:2009-06-20
  • 通讯作者: 吴建伟

Optimization of the method for solid phase extraction of antiviral peptides from haemolymph of diapausing larvae of Musca domestica with methanol.

  • Online:2009-06-20 Published:2009-06-20

摘要: 为了优化甲醇萃取滞育家蝇Musca domestica幼虫血淋巴中抗病毒肽的方法, 本实验分别比较甲醇及乙腈萃取血淋巴中抗病毒组分效果及最佳使用梯度、上样蛋白浓度、洗脱次数、洗脱流速及操作环境温度, 用Tricine-SDS-PAGE分析甲醇及乙腈的洗脱组分的构成。结果表明:甲醇萃取的活性组分多于乙腈萃取的; 用连续梯度(10%~100%)甲醇萃取的抗病毒活性组分, 比分别用10%, 30%, 50%, 70%, 90%和20%, 40%, 60%, 80%, 100%间断梯度洗脱的回收率高, 同时获得活性组分的重现性好。在0~4℃冰浴的环境下, 于250 mg/6 mL C18萃取柱内加入1.5±0.4 mg/mL蛋白浓度的血淋巴粗提液, 用1 mL/min流速4次洗脱, 抗病毒活性组分可以得到较好的分离效果。

关键词: 家蝇, 滞育, 血淋巴, 抗病毒肽, 固相萃取, 甲醇

Abstract: In order to optimize the conditions of antiviral peptide extraction from haemolymph of diapausing housefly (Musca domestica) larvae with methanol, we compared the separating results of methanol and methyl cyanide and selected the best extraction gradient. After studying the proper protein sample concentration, eluting times, eluting velocity and eluting temperature, we got a better protocol which leads to perfect results of antiviral peptide extraction. Tricine-SDS-PAGE results showed that continuous gradient methanol as an extractive organic solvent eluted more active fractions than methyl cyanide (P<0.05), and the continuous gradient elution with mthanol (10%-100%) was better than interrupted gradient (10%, 30%, 50%, 70% and 90%;20%, 40%, 60%, 80% and 100%, respectively)in the recovery rate and reproducibility of active fractions. Under 0-4℃, when 1.5±0.4 mg/mL haemolymph extracts was added into Sep-Pak C18 catridge and eluted 4 times with the flowing velocity of 1 mL/min, the antiviral fractions were separated better. Upon electrophoresis, only 2 target protein bands of active fractions were eluted by methyl cyanide, while more bands were eluted by methanol.

Key words: Musca domestica, diapause, hemolymph, antiviral peptide, solid phase extract, methanol