论文部分内容阅读
目的 鉴定朊蛋白 (PrP)基因转录启动子位置。方法 利用聚合酶链反应 (PCR)扩增人PrP基因外显子Ⅰ及其上游序列 ,序列鉴定后插入CAT报道质粒pBL CAT6 ,分别转染HeLa、COS7和Sh sy5y细胞系 ,检测CAT表达值 ;提取三种细胞蛋白 ,以条带移动实验检测细胞转录激活因子SP1含量。结果 人PrP基因外显子Ⅰ及其上游序列为GC富含 ,带有多个SP1可能性结合位点 ,但无明显TATA盒 ;瞬时转染结果显示这段序列可诱导 2～ 3倍的CAT表达增强 ;定量移动条带实验证明HeLa细胞中含有较高浓度的SP1,而COS7和Sh sy5 y细胞中SP1含量极低。结论 人PrP基因外显子Ⅰ及其上游序列具有启动子样功能 ,为弱的非TATA盒启动子 ;不同组织来源的细胞中SP1含量不同 ,在神经细胞系Sh sy5y中人PrP基因外显子Ⅰ及其上游序列的启动子活性似乎不依赖于SP1蛋白
Objective To identify the PrP gene transcriptional promoter location. Methods The exon Ⅰ and its upstream sequences of human PrP gene were amplified by polymerase chain reaction (PCR). After sequencing, CAT reporter plasmid pBL CAT6 was inserted into HeLa, COS7 and Sh5y5y cell lines to detect CAT expression. Three kinds of cell proteins were extracted, and the content of cell transcription activator SP1 was detected by band shift assay. Results The human PrP gene exon Ⅰ and its upstream sequence were GC-rich, with multiple SP1 binding sites but no obvious TATA box. Transient transfection results showed that this sequence could induce 2- to 3-fold CAT And the expression was enhanced. Quantitative mobile banding experiments showed that SP1 contained a higher concentration of SP1 in HeLa cells, while the SP1 content in COS7 and Sh5SyY cells was extremely low. Conclusions Exon 1 of human PrP gene and its upstream sequence have promoter-like function, which is a weak non-TATA box promoter. SP1 content in different tissue-derived cells is different. In human neuronal cell line Sh sy5y, the exon of human PrP gene The promoter activity of I and its upstream sequence appears to be independent of the SP1 protein