摘要
以人工合成的一对寡聚核苷酸为引物和人外周血淋巴细胞λＧＥＭ－１１基因文库为模板，通过ＰＣＲ扩增并克隆了人ＨＳＰ９０β基因上游－１１０２／＋６８ｂｐ片段。将该片段删切后克隆在报告基因──萤火虫荧光素酶基因５＇上游，转染Ｊｕｒｋａｔ细胞后测定不同刺激条件下细胞裂解液中的荧光酶活性并同时测定荧光酶ｍＲＮＡ水平。结果表明，ＨＳＰ９０β基因上游片段在正常生理状态下通过启动子介导较高水平的基础转录；热休克时该片段起负调控作用，主要的负调控区位于－５５４／－１７１ｂｐ之间，删除５＇远端上游序列后报告基因对丝裂原的反应性降低。
Using a pair of synthetic oligonucleotide as primers,human peripheral lymphocyte λGM-11 genomic DNA library as template,a fragment spanning-1102/+68bp of human heat shock protein 90βgene was amplified by polymerase chain reaction(PCR)and cloned into pGEM-4Z vector,The cloned fragment was truncated and ligated to an eukaryotic expression vector containing luciferase as a reporter gene. These subclones were transfected into Jurkat cells and the cellular luciferase activity and mRNA level were measured under either heat shock or PHA activation. Our results suggest that:(1)5' flanking sequencs of hsp90βmediates a relatively higher basal level expression via promotor region;(2)under heat shock,presence of the fragment surprisingly reduced luciferase activity,a heat shock induced negative regulatory element was restricted to-554/-171bp;and (3)PHA activation requires distal 5' sequence.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
1995年第4期241-249,共9页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金(新概念
新构思课题)
卫生部项目基金
