抗人Elp3的N末端抗体制备及在染色质免疫沉淀中的应用  

Preparation of Polyclonal Antibody Against N-terminal of Human Elp3 and Its Application in Chromatin Immunoprecipitation

作　　者：李芬[1] 田树娟[1] 许森[1] 武玉光[1] 孔艳[1] 张帅[1] 王艳芳[1] 

机构地区：[1]河南师范大学生命科学学院,新乡453007

出　　处：《中国生物化学与分子生物学报》2009年第10期919-925,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.30600343);河南省教育厅自然科学基金(No.2007180030);河南省重点学科建设项目资助~~

摘　　要：人Elp3(human elongator protein 3,hElp3)具有组蛋白乙酰转移酶活性,是与延伸中的RNA聚合酶Ⅱ结合的elongator复合物的催化亚基,可参与组蛋白的乙酰化修饰与基因的转录延伸.Elp3及其复合物功能异常与人类多种疾病相关.为运用染色质免疫沉淀等手段深入研究Elp3功能,PCR法克隆pYES2-hElp3质粒中编码hElp3的N端亲水区段(1～69氨基酸残基),构建原核表达载体pMXB10-hElp3-210,经IPTG诱导和几丁质柱纯化后,免疫兔制备多克隆抗体.ELISA检测显示,该抗体有较高的效价(不低于1∶2 500).免疫印迹实验结果表明,该抗体可与纯化的及HeLa细胞中的hElp3蛋白特异性结合.运用该抗体对转入elp3Δ菌株的人Elp3的染色质免疫沉淀实验结果表明,人Elp3可参与酵母SSA3基因的转录调控,这可能是人Elp3能够部分补偿酵母SSA3基因延迟表达缺陷的原因.Human elongator protein 3 （hElp3）, the catalytic subunit of the multi-subunit elongator complex associating with an RNA polymerase II holoenzyme engaged in transcriptional elongation, was first identified by its acetyhransferase activity, and subsequent data have provided further evidence that the enzyme is involved in histone acetylation and transcription. Variants of Elp3 gene are associated with many human diseases. In order to study the function of Elp3 by ehromatin immunoprecipitation （ChiP） and other techniques, we amplified the fragment of help3 gene encoding 69 amino acids in the N-terminal of yElp3 from pYES2-hElp3 template and inserted into the prokaryotie expression vector pMXB10 to construct the recombinant plasmid pMXB10-hElp3- N. After inducing the fusion protein expression in E. coli BL21 （DE3） by IPTG, a 36 kD intein-yElp3 fusion protein was obtained. Then the protein was purified by chitin affinity column and used to immune rabbits for acquiring antiserum. The results of ELISA and Western blot indicated that the polyelonal antibody was of high titration and specificity. ChIP assay with this antibody showed that help3 exerted the transcriptional regulatory function directly through its binding to the SSA3 promoter and ORF region in yeast, suggesting that it may rescue the delay activation of SSA3 in elp3A cells.

关 键 词：hElp3 原核表达 多克隆抗体 染色质免疫沉淀分析 

分 类 号：Q291[生物学—细胞生物学] Q78