凡纳滨对虾肌肉蛋白质双向电泳分离技术的建立及优化

Establishment and Optimization of Two-dimensional Electrophoresis(2-DE) Technique in Muscle Proteome of Litopenaeus vannamei

建立凡纳滨对虾肌肉组织双向电泳分离技术并对其进行优化。通过液氮研磨和液氮研磨＋超声破碎的方式提取对虾肌肉组织蛋白质。采用7 cm的IPG胶条进行双向电泳,凝胶染色后进行图谱分析,并对裂解液配方、胶条pH值、蛋白质上样量、凝胶染色进行优化。结果显示,以液氮研磨＋超声破碎,裂解液Ⅱ提取对虾肌肉蛋白质;采用pH 4-7 IPG胶条,180μg蛋白质上样量,并用考马斯亮蓝G-250胶体考染（蓝银）染色,成功得到背景清晰,分辨率较高的凡纳滨对虾肌肉蛋白质双向电泳图谱,这为凡纳滨对虾肌肉蛋白质组学分析及肌肉品质变化的分子标记识别提供基础。

Two-dimensional electrophoresis（2-DE） method on proteome of Litopenaeus vannamei was established and optimized. The shrimp proteins were extracted by grinding in liquid nitrogen and following by ultrasonication, separated by 2-DE with the 7 cm IPG strip. Image analysis software was applied to analyze the 2-DE images after staining. Effect of the lysis buffer, protein extraction method, pH of IPG gel strip, loading protein amount and stain method on 2-DE images were detected. The results showed that the 2-DE pattern of shrimp protein with clear background and better resolution was successfully obtained using lysis buffer Ⅱ, grinding in liquid nitrogen and following by ultrasonication, 7 cm pH 4-7 IPG gel strips, loading the 180 μg protein and dying the gels stained by blue silver. The study would provide a technical reference for the proteomic analysis and molecular marker identification of Litopenaeus vannamei muscle.