黄鳍鲷骨骼肌伴肌动蛋白的纯化及其抗体制备

CORC > 集美大学

	黄鳍鲷骨骼肌伴肌动蛋白的纯化及其抗体制备
	杜雪莉 ; 周利亘 ; 王锡昌 ; 熊何健 ; 苏文金 ; 曹敏杰
刊名	http://epub.edu.cnki.net/grid2008/brief/detailj.aspx?filename=sckx200803019&dbcode=CJFQ&dbname=CJFQ2008
	2012-06-05 ; 2012-06-05
关键词	黄鳍鲷伴肌动蛋白纯化抗体 Sparus latus nebulin purification antibody S917.4
其他题名	Purification and antibody preparation of nebulin from the skeletal muscle of sea bream(Sparus latus)
中文摘要	采用Sephacryl S-400凝胶过滤柱层析和电洗脱等纯化方法,首次从黄鳍鲷骨骼肌中分离纯化到伴肌动蛋白,并制备了特异性多克隆抗体。多克隆抗体经Protein A-Sepharose亲和层析柱纯化得到高纯度免疫球蛋白G(IgG)。采用聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫斑点印迹(Dot-Blot)和免疫印迹(Western-Blot)等方法对纯化的伴肌动蛋白及其多克隆抗体进行分析鉴定,并研究了不同贮藏条件下伴肌动蛋白的变化情况。SDS-PAGE结果显示本研究从黄鳍鲷骨骼肌中分离纯化了高纯度伴肌动蛋白;Dot-Blot检测结果显示兔抗黄鳍鲷伴肌动蛋白多克隆抗体效价为5×104;Western-blot检测表明兔抗黄鳍鲷伴肌动蛋白多克隆抗体能与肌原纤维蛋白中的伴肌动蛋白发生特异性反应,而与其它蛋白不发生免疫交叉反应。; As a giant actin-binding protein with molecular weight of approximately 500-900 ku,nebulin is thought to act as a molecular template that regulates the length of thin filaments and plays an essential role in the assembly and maintenance of I-Z-I bands in myofibrillar muscles.Though nebulins from mammals have been studied widely,much less study was performed on nebulin from fish muscle.In the present study,nebulin from the skeletal muscle of sea bream(Sparus latus) was first extracted with low ionic strength buffer,followed by applying to Sephacryl S-400 gel filtration chromatography and finally purified by electrophoretic elution.Purified nebulin revealed a single protein band on sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE),suggesting its high homogeneity.A polyclonal rabbit anti-nebulin antibody was thus prepared by immunizing rabbit with purified nebulin and immunoglobulin G(IgG) was purified by Protein A-Sepharose affinity column.The immunogeneicity of the purified antibody was further identified by Dot-Blot and Western-Blot.The potency of anti-nebulin antibody reached 5×104 by Dot-Blot analysis.Western-Blot revealed that the antibody immunologically reacted with purified nebulin and nebulin in myofibrils while no cross reaction with other myofibrillar proteins was detected,suggesting the high specificity of the polyclonal antibody.Thus,the antibody prepared in the present study will surely be beneficial to further study concerning the detection of this protein immunologically.The degradation of nebulin during fish storage at two temperatures of 4℃ and 18℃ was also investigated by SDS-PAGE.At 4℃,protein degradation could be detected after storage for 9 d and after 18 d,more than 70% of the nebulin original band disappeared.However,at 18℃,nebulin deterioration could be detected even at 24 h and after 120 h,the original protein band disappeared completely.The decomposition of nebulin as detected by SDS-PAGE was in accordance with sensory freshness change,suggesting the completeness of nebulin is an index of fish muscle freshness.Interestingly,the largest protein(titin) almost did not change even after storage at 4℃ for 18 d or at 18℃ for 188 h.In conclusion,this present study,especially the anti-nebulin antibody prepared provided an effective tool for the investigation of fish freshness.; 【作者单位】集美大学生物工程学院；浙江省农业科学院食品加工研究所；上海水产大学食品学院；集美大学生物工程学院福建厦门361021；上海200090；浙江杭州310021；福建厦门361021；【作者英文名】DU Xue-li1,2,ZHOU Li-gen3,WANG Xi-chang2,XIONG He-jian1,SU Wen-jin1,CAO Min-jie1(1.School of Biotechnology,Jimei University,Xiamen 361021,China;2.College of Food Science and Technology,Shanghai Fisheries University,Shanghai 200090,China;3.Institute o
语种	中文
内容类型	期刊论文
源URL	[http://ir.calis.edu.cn/hdl/235041/18446]
专题	集美大学
推荐引用方式 GB/T 7714	杜雪莉,周利亘,王锡昌,等. 黄鳍鲷骨骼肌伴肌动蛋白的纯化及其抗体制备[J]. http://epub.edu.cnki.net/grid2008/brief/detailj.aspx?filename=sckx200803019&dbcode=CJFQ&dbname=CJFQ2008,2012, 2012.
APA	杜雪莉,周利亘,王锡昌,熊何健,苏文金,&曹敏杰.(2012).黄鳍鲷骨骼肌伴肌动蛋白的纯化及其抗体制备.http://epub.edu.cnki.net/grid2008/brief/detailj.aspx?filename=sckx200803019&dbcode=CJFQ&dbname=CJFQ2008.
MLA	杜雪莉,et al."黄鳍鲷骨骼肌伴肌动蛋白的纯化及其抗体制备".http://epub.edu.cnki.net/grid2008/brief/detailj.aspx?filename=sckx200803019&dbcode=CJFQ&dbname=CJFQ2008 (2012).