不同细胞破碎方法对肉牛、牦牛背最长肌双向电泳图谱的影响

Comparison of Different Cell Disruption Methods for Two-Dimensional Electrophoresis Analysis of Beef and Yak Muscle

采用液氮研磨、液氮研磨辅助超声波破碎和液氮研磨辅助玻璃珠破碎3种细胞破碎方法对肉牛、牦牛背最长肌蛋白质提取和双向电泳图谱效果的最佳方法进行研究。在相同裂解液组成、等电聚焦程序、十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electropheresis,SDS-PAGE)操作、银染法染色等双向电泳条件的处理下,通过Bradford法分别测定不同方法提取的肉牛和牦牛肉背最长肌中蛋白质的含量,并采用PDQuest 8.0.1软件对双向电泳图谱的效果进行分析。结果表明:3种细胞破碎方法提取的肉牛和牦牛背最长肌蛋白质含量差异显著,其中液氮研磨辅助超声波破碎法((2.59±0.15)、(2.84±0.29)μg/μL)显著高于其他2种方法,液氮研磨辅助玻璃珠破碎法((2.01±0.04)、(2.21±0.02)μg/μL)显著高于液氮研磨法((1.77±0.11)、(1.95±0.19)μg/μL)。3种方法所得肉牛背最长肌双向电泳图谱的蛋白点个数分别为(279±19)、(581±15)、(363±21)个;所得牦牛肉双向电泳图谱的蛋白点个数分别为(292±15)、(596±12)、(385±17)个。3种方法中液氮研磨辅助超声波破碎法提取的肉牛、牦牛背最长肌蛋白质含量最高,数量最多,蛋白点分离程度最好,并且3种细胞破碎方法在牦牛背最长肌中提取的蛋白质含量与肉牛背最长肌中提取的蛋白质含量差异不显著。

Three cell disruption methods, i.e., liquid nitrogen grounding, liquid nitrogen grounding aided ultrasonication and liquid nitrogen grounding with glass beads were investigated to find the most appropriate one for the extraction and twodimensional electrophoresis analysis of proteins in longissimus dorsi muscles of beef cattle and yak. The same lysis buffer was used in the different cell disruption methods and the two-dimensional electrophoresis analysis was performed using isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electropheresis（SDS-PAGE） by silver staining. The protein contents of longissimus dorsi muscles were determined by the Bradford method. Analysis of the two-dimensional electrophoresis images was conducted using using PDQuest 8.0.1 software. Results showed that the protein contents of beef and yak longissimus dorsi muscles with the three cell disruption methods were significantly different,（2.59 ± 0.15） and（2.84 ± 0.29） μg/μL,（2.01 ± 0.04） and（2.21 ± 0.02） μg/μL, and（1.77 ± 0.11） and（1.95 ± 0.19） μg/μL for liquid nitrogen grounding aided ultrasonication, liquid nitrogen grounding with glass beads and liquid nitrogen grounding, respectively. The numbers of protein spots in the electrophoresis images of beef longissimus dorsi obtained using these three cell disruption methods were（279 ± 19）,（581 ± 15） and（363 ± 21）, respectively, whereas those of yak longissimus dorsi were（292±15）,（596 ± 12） and（385 ± 17）, respectively. Among these three methods, liquid nitrogen grounding aided ultrasonication yielded the highest protein contents, the greatest number of proteins and the best separation of protein spots from beef and yak longissimus dorsi. No significant differences in the protein contents of beef and yak longissimus dorsi were observed among the three cell disruption methods.