牛冻、鲜精子差异蛋白的双向电泳和质谱鉴定的初步研究  

Primary Study on Identify of the Differential Proteins between Bovine Frozen and Fresh Spermatozoa Using 2-DE and MS

作　　者：吴承疆[1,2] 朱化彬[2] 王栋[2] 郝海生[2] 杜卫华[2] 赵学明[2] 路永强[3] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]北京市畜牧兽医总站,北京100107

出　　处：《中国畜牧兽医》2010年第11期100-105,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金资助项目(30671502);国家"863"高技术研究发展规划(2006AA10Z143)

摘　　要：采用适合于精子裂解的热Trizol法制备牛精子全蛋白质,应用线性固相IPG胶条(pH 3～7、24cm)进行精子全蛋白2-DE电泳,在冻精和鲜精蛋白图谱中分别检测到839±34个蛋白点和564±16个蛋白点,经差异比较和显著性检验,在冻、鲜精子中共得到19个具有显著差异的蛋白点,在冻精中表达上调的蛋白点有9个,表达下调的蛋白点有1个,只在冻精中表达的蛋白点有9个。经过酶解和质谱鉴定,获得了3个差异蛋白点的质谱信息,生物信息学分析结果发现,这3个差异点分别为中性鞘磷脂酶激活结合因子(FAN)、谷胱甘肽S转移酶(GST-Mu5)及锌离子结合细胞色素氧化酶,分别与细胞应激和凋亡有关。推测认为,这3种与细胞应激反应及凋亡相关的蛋白在冻精中显著增高可能与精子冷冻损伤有关。Heat Trizol method was used to prepare the global protein of bovine spermatozoa,and the global proteins of fresh sperm and frozen sperm was separated by the technique of 2-DE with linear IPG（pH 3to pH 7,24cm）.839±34protein spots were obtained from the sample of frozen spermatozoa,while 564±16from the sample of fresh spermatozoa.19differenti-al proteins between frozen and fresh sperm were detected after analysis and significance test with software of ImageMasterTM2D Platinum,9of which were expressed up-regulated while one of which was down-regulated in the sample of frozen spermatozoa.In addition,9spots were found only in frozen spermatozoa.After enzymolysis and MS detection,mass spectrum information of 3differential proteins were obtained,and the differential proteins were considered to be neutral sphingomyelinase（N-SMase） activation associated factor（FAN）,glutathione-S-transferase（Mu5）and zinc ion binding structure of bovine heart cytochrome c oxidase in the fully oxidized state,which were related to cell stress and apoptosis.It was presumed that the increasing of these proteins significantly in frozen spermatozoa were involved with cryodamage of spermatozoa.

关 键 词：冻、鲜精子 2-DE 质谱分析 冷冻损伤 

分 类 号：TQ937[化学工程]