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作 者:刘冰[1] 王宇祥[1] 石慧[1] 王启贵[1] 李辉[1]
机构地区:[1]东北农业大学动物科学技术学院,哈尔滨150030
出 处:《农业生物技术学报》2009年第1期47-51,共5页Journal of Agricultural Biotechnology
基 金:国家自然科学基金重点项目(No.30430510);国家重点基础研究发展规划(973)(No.2006CB102105);黑龙江省自然科学基金重点项目(No.ZJN0604-01)资助
摘 要:制备鸡(Gallus gallus)CAAT增强子结合蛋白α(CAAT/enhancer-binding proteinα,C/EBPα)的多克隆抗血清,并分析其组织表达特性。通过限制性酶切,从T-C/EBPα质粒中获得C/EBPα基因的编码区序列(coding sequence,CDS),构建原核表达载体,以大肠杆菌(Escherichia coli)为宿主,用IPTG诱导重组pGEX6p/C/EBPα的表达,经Glutathione Sepharose 4B柱层析纯化后,以其为免疫原制备兔抗鸡C/EBPα多抗。ELISA方法测定抗体效价达1∶12800,Western blot方法显示抗体具有较高的特异性。用此抗血清分析了鸡C/EBPα的组织表达特性,结果表明,鸡的回肠、肾脏、肌胃、腺胃、心脏、肝脏、腹脂、脾脏等组织中C/EBPα蛋白表达量较高,而在胸肌、腿肌中表达量较低。同时分析C/EBPα基因在高低脂系公鸡肝脏组织中的表达差异,结果显示该基因在低脂系公鸡肝脏中表达量较高。The coding sequence (CDS) of CAAT/enhancer-binding protein et (C/EBPα) gene was obtained from T-C/EBPα plas-mid by enzyme digestion. The recombinant C/EBPα was expressed in Eschenchia coli with 1PTG induction and purified by Glutathione Sepharose 4B affinity chromatography. The purified C/EBPα was used as immunogen to immunize the rabbit. The titer of the generated antiserum was detected to be 1:12800 by ELISA. Western blotting result showed that the antiserum had high specificity. Expression characteristics showed that C/EBPα expressed abundantly in muscle stomach, glandulose stomach, kidney, intestine, liver, heart, fat, spleen and lung respectively while little in muscle. The antiserum was meanwhile used to analyze the C/EBPα expressior characteristics between fat and lean line roosters, showing that C/EBPα expressed higher in lean line rooster livers than in fat ones.
关 键 词:鸡 CAAT增强子结合蛋白α 抗血清 表达特性
分 类 号:S188[农业科学—农业基础科学]
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