超低温保存对猪精子酶蛋白的影响

Effect of ultra-low temperature storage on zymoprotein of porcine sperm

为研究猪精子超低温保存前后差异酶蛋白表达,以期在蛋白质水平探索猪精子冷冻损伤机制,本研究采用双向电泳分离精子蛋白,硝酸银染色凝胶,ImageMaster2D platinum 5.0软件分析差异蛋白质斑点并经MALDI Tof/Tof质谱鉴定。结果表明:冷冻前后精子存在53个差异蛋白点,在NCBInr蛋白数据库中搜索到有意义的差异酶蛋白6种:类组氨酸磷酸酶14(like-PHP14)、烯醇酶(ENO)、二氢硫辛酸脱氢酶(PDC3)、谷胱甘肽过氧化物酶(GSH-Px)、鸟苷酸环化酶受体和RNA聚合酶II相关蛋白1,前4种冷冻-解冻后下调,后2种在冷冻-解冻精子中未检测到。说明冷冻前后猪精子存在差异酶蛋白质,为进一步研究猪精子蛋白质组学及揭示精子冷冻损伤机制提供了依据。

In order to study the differential zymoprotein expression before and after ultra-low temperature storage of porcine sperm and further explore the mechanism of sperm cryodamage,the protein change was investigated by means of separating the sperm protein by two-dimensional electrophoresis,staining gel with silver nitrate and analyzing the differential protein spots by the software ImageMaster 2D platinum 5.0 and through MALDI Tof/Tof mass spectrometry.The results showed that there were 53 differential protein spots between the fresh and frozen-thawed sperms.In the NCBInr database there were 6 differential zymoproteins searched：like 14 kD histidine phosphatase（likePHP14）,enolase（ENO）,dihydrolipoyl dehydrogenase（PDC3）,glutathion peroxidase（GSH-Px）,guanylyl cyclase receptor and RNA polymerase II associated protein 1,and after freeze-thaw,the likePHP14,ENO,PDC3 and GSH-Px were reduced and the rest were undetected.It was concluded that there were differential zymoproteins between the fresh and frozen-thawed sperms,giving a basis for further researching into porcine sperm proteomic and revealing the cryodamage mechanism of porcine sperm.