中国明对虾肌肉组织蛋白质双向电泳技术体系的建立  

Establishment of two-dimensional electrophoresis(2-DE) technique in muscle proteome of Fenneropenaeus chinensis

作　　者：刘志远[1] 励建荣[2] 李学鹏[2] 李婷婷[3] 王彦波[1] 黄和[4] 陈华健 陆维克 

机构地区：[1]浙江工商大学食品与生物工程学院,浙江杭州310012 [2]渤海大学化学化工与食品安全学院,食品科学研究院,辽宁锦州121013 [3]大连民族学院生命科学学院,辽宁大连116600 [4]广东海洋大学食品科技学院,广东湛江524088 [5]湛江国联水产开发股份有限公司,广东湛江524022 [6]杭州博拓生物技术有限公司,浙江杭州311121

出　　处：《水产学报》2013年第2期288-296,共9页Journal of Fisheries of China

基　　金：国家“十二五”科技支撑计划(2012BAD29B06);浙江省杰出青年科学基金项目(R3110345);辽宁省食品安全重点实验室暨辽宁省高校重大科技平台开放课题(LNSAKF2011029)

摘　　要：为了探索并建立中国明对虾的肌肉蛋白双向电泳体系,实验将中国明对虾的肌肉组织溶解处理后,通过固相pH梯度胶条等电聚焦、SDS-PAGE垂直电泳对蛋白质进行分离,对不同的裂解液配方、IPG胶条的pH范围及其长度、等电聚焦程序、上样量等进行了优化,并分别利用银染和考染方法进行染色,应用PDQuest软件对图谱进行了初步分析。结果显示,中国明对虾肌肉蛋白的等电点主要位于4～7之间,裂解液中添加硫脲、CHAPS、DTT等,可以增加对虾肌肉蛋白的提取率;采用17 cm,pH 4～7的中型IPG胶条,主动水化上样120μg,适当延长除盐时间和等电聚焦时间,搭建盐桥,采用浓度为12.5%的二向分离胶和硝酸银染色,能有效提高双向电泳(2-DE)图谱中蛋白点的分离度和分辨率;建立的中国明对虾肌肉蛋白双向电泳体系具有良好的重复性和稳定性。In order to use two-dimensional electrophoresis（2-DE）analysis in the muscle proteins ot tghlnese shrimp （Fenneropenaeus chinensis）, its muscle proteins were separated using immobilized pH gradient 2-DE after dissolving. By optimizing different extraction methods, pH of IPG gel strips and its length, isoelectric focusing programs, and loading amount, etc, the proteins were successfully extracted from Chinese shrimp muscle and were separated by 2-DE. After silver staining or Coomassie brilliant blue staining, PDQuest image analysis software was applied to analyze the 2-DE images. The results showed that most of shrimp muscle protein isoelectric points were between 4 and 7. The 2-DE related techniques was constructed and optimized in muscle proteome of Chinese shrimp by comparative tests on different extraction methods,IPG gel strips, isoelectric focusing programs, salt bridge, and sample volume, etc. The results showed that the resolution and reproducibility of 2-DE profiles were significantly improved by adding thiourea, CHAPS and DTT in lysis buffer, active rehydrating of 17 cm （ pH 4 - 7 ） IPG gel strips, loading the sample 120 p,g, prolonging the time of desalting,increasing the voltage and power of isoelectric focusing, employing the salt bridge, preparing 12.5% SDS-PAGE gel,and dying the gels by the silver staining. It shows that the repeatability and stability are good enough.

关 键 词：中国明对虾 肌肉蛋白质 双向电泳 蛋白质组学 

分 类 号：S917[农业科学—水产科学]