p65 DNA结合域的克隆、表达及其酵母双杂交自身激活作用检测  

Cloning and expression of NF-κB p65 subunit DNA binding domain and detection of its self-activation in the yeast two-hybrid system

作　　者：徐祥[1] 梁华平[1] 刘昕[2] 代佳平[2] 刘琛[1] 罗艳[1] 王正国[1] 

机构地区：[1]第三军医大学附属大坪医院野战外科研究所第一研究室,重庆400042 [2]第三军医大学基础医学部分子遗传学教研室,重庆400038

出　　处：《第三军医大学学报》2003年第2期123-126,共4页Journal of Third Military Medical University

基　　金：国家重点基础研究发展规划资助项目 ("973"项目 ) (G19990 54 2 0 3) ;国家自然科学基金资助项目 ( 30 0 80 0 0 9) ;重庆市科委基金资助项目 ( 2 0 0 0 6 319)

摘　　要：目的　获得NF κBp65亚基DNA结合域cDNA及构建酵母双杂交系统中的靶基因 ,并检测靶基因的表达和自身激活活性。方法　利用引物二聚体搭桥技术 ,扩增p65亚基DNA结合域基因片段 ,并将其克隆入酵母双杂交载体pG BKT7DNA BD。应用醋酸锂法转化酵母细胞AH10 9,在SD Trp选择培养基上培养 ,观察转化株的生长情况。按尿素 SDS法抽提酵母蛋白 ,用SDS PAGE电泳和Western blot鉴定靶蛋白在酵母细胞中的表达。利用液相法和平板法检测阳性转化株 β 半乳糖苷酶的活性。 结果　获得p65DNA结合域基因片段 ,并成功构建酵母双杂交系统中的靶基因。靶基因能在酵母细胞中表达 ,对酵母细胞无毒性作用 ,无自身激活活性。结论　NF κBp65亚基DNA结合域可作为酵母双杂合系统中的靶基因 ,用于肽库筛选 。Objective To obtain NF κB p65 subunit DNA binding domain cDNA, and measure its autonomous activation in yeast two hybrid system. Methods After the fragment of NF κB p65 subunit DNA binding domain was amplified with primer dimer bridge building, it was inserted into pGBKT7 DNA BD vector (named as pGBKT7 DNA BD/p65 ). After being automatically sequenced, the recombinant plasmid was transformed into yeast cells AH109 with polyethylene glycol/lithium acetate method, and the growth of transformants were observed in the SD/ Trp selective medium. After the yeast total protein was extracted by using Urea/SDS method, the expression of pGBKT7 DNA BD/p65 in the yeast cells was identified with SDS PAGE and western blotting. β galactosidase activity of positive clones was tested with liquid assay and plate assay respectively. Results NF κB p65 subunit DNA binding domain cDNA was obtained, and inserted into pGBKT7 DNA BD vector successfully. NF κB p65 subunit DNA binding domain was expressed in the AH109 cells, but had neither ability of autonomous reporter gene activation, nor toxicity to the yeast cells. Conclusion NF κB p65 subunit DNA binding domain can serve as target gene of yeast two hybrid system used in screening of polypeptide library to trap the protein interacting with it.

关 键 词：P65 DNA结合域 克隆 核因子ΚB 酵母双杂交 自身激活作用 酵母表达 炎症反应 

分 类 号：Q785[生物学—分子生物学] R364.5[医药卫生—病理学]