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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250mg/6mLC18萃取柱内加入1.5±0.4mg/mL蛋白浓度的血淋巴粗提液,用1mL/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。
In order to optimize the method of methanol extraction of antiviral peptides in the hemolymph of larvae of Musca domestica, the effects of methanol and acetonitrile on the antiviral components in the haemolymph were studied. The best gradient, sample concentration and elution times , Elution flow rate and operating ambient temperature, the composition of the eluted fractions of methanol and acetonitrile was analyzed by Tricine-SDS-PAGE. The results showed that the active components extracted by methanol were more than those extracted by acetonitrile. The antiviral active components extracted by methanol with a continuous gradient (10% -100%) were more effective than those by 10%, 30%, 50%, 70%, 90 % And 20%, 40%, 60%, 80%, 100% intermittent gradient elution recovery rate, while obtaining good reproducibility of the active ingredient. Under the environment of 0 ~ 4 ℃ ice bath, the hemolymph crude extract with protein concentration of 1.5 ± 0.4mg / mL was added into the 250mg / 6mLC18 extraction column and eluted 4 times with the flow rate of 1mL / min, the antiviral active components could be Get better separation effect.