一种适合稻水象甲双向电泳的蛋白质提取方法

A protein extraction method suitable for 2-DE analysis of rice water weevil

【背景】蛋白样品制备是2-DE蛋白组学研究的关键步骤,无杂质、高纯度的蛋白样品是获得良好的2-DE结果的基础。选择合适于昆虫蛋白组学研究的常规蛋白提取方法是2-DE研究的前提。【方法】通过饱和酚法和TCA-丙酮法提取稻水象甲成虫总蛋白,比较2种方法的优缺点,以回避昆虫组织蛋白提取时可能出现的问题。【结果】TCA-丙酮法的蛋白质产率(14.55μg·mg-1)显著高于饱和酚法(9.30μg·mg-1),2种方法获得的SDS-PAGE条带结果相近,且高丰度表达蛋白在TCA-丙酮中更高表达;饱和酚的2-DE图谱蛋白点清晰,TCA-丙酮法2-DE图谱蛋白点规则但背景模糊,出现多条横纹。【结论与意义】饱和酚法适合稻水象甲成虫蛋白组学的研究。这2种方法也能适用于其他昆虫,但不同的昆虫体内有不同的干扰组分来干扰蛋白质的提取,所以获得的蛋白纯度及提取得率可能并不一样。试验结果说明了2种蛋白提取方法对2-DE研究的影响,并为稻水象甲成虫的蛋白组学研究提供了一定的参考依据。

[ Background] Protein sample preparation is a central step in a 2-DE proteomics as pure protein samples are necessary. There is a need to establish a routine protocol for the application of proteomics analysis in insects. [ Method] This study focused on the specific protein extraction problems in insect tissned and evaluated two protocols to bypass them. The methods of phenol extrac- tion （PE） and TCA/acetone precipitation （TCA） were evaluated for proteins isolation from adult rice water weevil, Lissorhoptrus oryzophilus Kuschel. [ Result] TCA method yielded a greater protein extraction rate （ 14.55μg·mg-1 ） than the PE protocols （9.30μg·mg-1）. The two methods got similar SDS-PAGE results but the expression of protein was higher in TCA than PE; For 2-DE, the PE protocol was optimal, resulting in good background and clear protein spots and TCA had an indistinct background and oc- curred a few bands were present. [ Conclusion and significance] Among the applied methods, PE precipitation extraction was found to be more suitable than TCA for rice water weevil proteomic analysis. These protocols should also be applicable to other insects. Dif- ferent insects can vary considerably in the amounts and types of interfering compounds they produce, so the results of protein extrac- tion rate and purity in different insects should be assessed. The study proves that various protein extraction methods result in different protein rates and 2-DE products. It provides basic protocols for proteomic analysis of adult rice water weevil.