基因ifi56在全反式维甲酸诱导急性早幼粒细胞白血病细胞分化中的表达及其真核表达质粒的构建  

Expression of ifi56 Gene in ATRA-induced APL Cell Differentiation and Construction of ifi56 Gene Eukaryotic Expression Plasmid

作　　者：张长林[1,2] 许桂平[1] 肖澍[1] 李冬[1] 贾培敏[1] 童建华[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院上海血液学研究所,上海200025 [2]南昌大学第一附属医院检验科,江西南昌300006

出　　处：《中国实验血液学杂志》2010年第5期1159-1162,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30670882和30971275);国家863计划(编号2006AA02Z19A);南昌大学科技基金项目(张长林)

摘　　要：本研究探讨ifi56基因在全反式维甲酸诱导急性早幼粒细胞白血病(APL)细胞分化中的表达,构建人ifi56真核表达载体并证实融合蛋白在细胞内表达及定位情况。用RT-PCR技术检测APL细胞NB4经维甲酸处理不同时间后ifi56的表达,并从白血病细胞NB4中扩增ifi56全长编码序列,将其亚克隆到真核表达载体pEGFP-C1中,转染到293T细胞中,提取细胞蛋白,用Western blot方法检测蛋白表达情况,荧光显微镜检测IFI56的定位。结果表明,ifi56在未经处理的NB4细胞中几乎检测不到,当用维甲酸处理72小时后明显升高,并成功将ifi56插入到质粒pEGFP-C1中,Western blot检测到融合蛋白表达,分子量约为83 kD。IFI56重组蛋白在293T细胞内主要定位于细胞胞浆中。结论:ifi56表达随着APL细胞分化明显升高,成功构建ifi56基因真核表达载体,IFI56蛋白主要定位于细胞浆。This study was purposed to investigate the expression of ifi56 gene in the ATRA-induced acute promyelocytic leukemia（APL） NB4 cell differentiation and to construct the eukaryotic expression plasmid of ifi56 gene.RT-PCR was used to detect the expression of ifi56 in NB4 cells treated with ATRA for different time.Human ifi56 cDNA was amplified by RT-PCR and cloned into pEGFP-C1 vector,then was transfected into 293T cells.The expression of the recombinant protein in 293T cells was detected by Western blot.The localization of IFI56 protein was observed by fluorescence microscopy.The results showed that the ifi56 mRNA was almost undetectable in untreated NB4 cells,but it signi-ficantly increased after ATRA treatment for 72 hours.The cDNA fragment of ifi56 was inserted into the expressing plasmid pEGFP-C1 successfully.The expression of EGFP-IFI56 fusion protein with a molecular weight about 83 kD was detected by Western blot.The EGFP-IFI56 protein was localized in cytoplasm mainly.It is concluded that the expression of ifi56 is enhanced significantly when the differentiation of APL cells was induced by ATRA.Gene ifi56 is successfully cloned into eukaryotic expression vector and the fusion protein is expressed in the cytoplasm mainly.

关 键 词：ifi56基因 全反式维甲酸 急性早幼粒细胞白血病 真核表达质粒构建 

分 类 号：R733.71[医药卫生—肿瘤] Q789[医药卫生—临床医学]