摘要
本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针，从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆，经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析，证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析，鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中，并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失，构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后，以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达，结果发现在ＣＤ２基因５’上游具有很弱的启动子活性，初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。
In order to find the expected promoter element that may exist as an astypical sequence in the 5' flanking sequence of the human T lymphocyte CD2 gene, we used a cDNA fragment derived from the 5' portion of human CD2 cDNA as the probe to screen a human T lymphocyte genormic library. Two overlapping lambdaGEM recombinant clones were successively isolated. Restriction enzyme digestion and hybridization analysis showed that one of clones contained the 5' flanking sequence of the CD2 gene. A 2.5kb fragment which contains two DNase I hypersensitive sites and the transcriptional starting point of the 5' flanking sequence and its mutants were subcloned into an expression vector pMG3 upstream to a luciferase reporter gene. Transient expression assay revealed the presence of a low promoter activity within this fragment (-1.2kb～-9.8bp), suggesting that a weak promoter, similar to other promoters of T cell surface antigen genes. was found in the 5' upstream of the CD2 gene.
基金
国家自然科学基金
关键词
CD2启动子
淋巴细胞
克隆
鉴定
CD2 promoter
Human T lymphocytes
Luciferase assay
