斑节对虾血淋巴低丰度蛋白双向电泳技术体系的优化

Optimation of two-dimensional electrophoresis(2-DE) technique in low abundance hemolymph proteome of Penaeus monodon

文章探索并优化了斑节对虾（Penaeus monodon）血淋巴双向电泳技术体系，对斑节对虾血淋巴总蛋白抽提方法进行优化，减小高丰度蛋白的影响；对双向电泳过程各步骤，如IPG胶条pH范围的选择、等电聚焦程序、上样量等进行了优化，得到高分辨率的2-DE图谱，用PDQuest软件进行了初步分析。结果表明，用PEG 6000能更快捷有效地去除血淋巴中的高丰度蛋白，增加低丰度蛋白的点数及浓度；在抽提蛋白过程中，利用预冷的80%丙酮洗涤，在一向电泳过程中设置100 V低压除盐，能更有效地去除蛋白中的盐分；采用18 cm、pH 3~10 NL 的中型胶条，能更好地显示低丰度蛋白点的分布；被动水化上样300 μg结合硝酸银染色方法，能有效提高双向电泳图谱中蛋白点的分离度和分辨率。该试验为斑节对虾及其他甲壳动物血淋巴蛋白质组学相关研究提供了稳定可重复的研究方法。

We optimized the two-dimensional electmphoresis technique for analyzing hemolymph in Penaeus monodon. To obtain high resolution 2-DE patterns, we examined the protein extraction, IPG-strip length, separation scope of IPG strip, sample loading amount and focusing condition by PDQuest software. Results indicate that PEG 6000 removed high abundance protein in haemolymph faster and more effectively, and increased the concentration of low abundance protein. The protein preparation with 80% TCA-acetone extraction treatment in isoelectric focusing was effective. The optimum 2-DE patterns in the proteins of Penaeus monodon were successfully separated by using pH 3~10 NL IPG strips with 18 cm length and 300 μg protein sample. The study provides stable and replicable method for relevant studies on hemolymph proteame of P.monodon and other similar crustaceans.