免疫共沉淀检测人P65和SMRT蛋白间相互作用.docVIP

免疫共沉淀检测人P65和SMRT蛋白间相互作用 王春美1， 盛光耀1， 卢洁2， 邹湘1， 方营旗1， 白松婷1， 郝庆飞1， 谢 垒1 （郑州大学：1郑州大学第一附属医院儿科，2郑州大学公共卫生学院流行病学与统计学教研室，郑州 450052） 摘要：目的 使用免疫共沉淀技术检测人P65和SMRT蛋白在儿童急性髓细胞白血病（AML）细胞株THP-1中相互作用。方法 分别构建带有HA标签和Myc标签的人P65和SMRT蛋白的重组真核表达载体pCMV-HA-p65和pCMV-Myc-SMRT，测序、酶切鉴定正确后，共转染THP-1细胞，免疫共沉淀检测二者间相互作用。结果 成功构建了人P65和SMRT蛋白真核表达载体pCMV-HA-p65和pCMV-Myc-SMRT，转染THP-1细胞，抗HA和Myc单克隆抗体分别沉淀HA-p65和Myc-SMRT相互作用蛋白复合物后，用抗Myc和HA单克隆抗体分别进行Western Blotting检测，可以检测到HA-p65和Myc-SMRT蛋白的表达。结论 成功构建了人P65和SMRT蛋白重组真核表达载体，在THP-1细胞株中表达后，免疫共沉淀证实P65和SMRT蛋白间存在相互作用，为进一步探讨儿童AML发病机制和白血病的靶向治疗提供理论依据。 关键词：免疫共沉淀， P65，SMRT，髓细胞白血病 Identification of the Interaction Between P65 and SMRT by Co-Immunoprecipitation Wang Chun-mei1, Sheng Guan-gyao1, Lu Jie 2, Zou Xiang1, Fang Ying-qi1,Bai Song-ting1,Hao Qing-fei1, Xie Lei1 ( Department of Paediatrics, the First Affiliated Hospital, Department of Epidemiology and Hygiene Statistics, School of Punlic Health, Zhengzhou University, Zhengzhou 450052) Abstract: Objective To identify the interaction between P65 and SMRT proteins in acute myelocytic leukemia cell lines(THP-1) by co-immunoprecipitation. Methods 基金项目：河南省医学科技攻关项目（2008） Supported by the Province Medical Science and Technique Foundation of Henan, China(2008) 作者简介：王春美（1983-），女，河南省驻马店人，博士研究生，主要从事儿科血液病和肿瘤疾病的研究。电话E-mail:meichun123@126.com 通讯作者：盛光耀，电话(0371E-mail：shenggy666@ The recombinant eukaryotic expression vectors of pCMV-HA-p65 and pCMV-Myc-SMRT were constructed in vitro, after restriction enzyme digestion and DNA sequenced, then co-transfected in THP-1 cell lines. The interaction between P65 and SMRT were detected by co-immunoprecipitation. Results Restriction enzyme digestion and DNA sequencing showed that the recombinant eukaryotic expression vectors of pCMV-HA-p65 and pCMV-Myc-SMRT were constructed correctly. After immunoprecipitated by anti-HA antibody and anti-Myc antibody，HA-p65and Myc-SMRT were identified by Western blotting with anti-Myc antibody and anti-HA antibody from immun0precipitated com