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目的:分析人β1整合素在HaCat细胞中核心启动片段。方法:以本室构建的含整合素β1全长启动子1745bp基因序列(-1745bp～+11bp)的重组载体pGL3-1756为模板,用含有酶切位点的特异性引物扩增出整合素β1启动子-845bp～-304bp间基因序列,克隆至荧光素酶表达载体pGL3basic,构建含正确目的基因重组载体pGL3-542。用pGL3-1756、和pGL3-542质粒转染人表皮细胞株HaCat进行活性分析。结果:酶切及序列测定表明,克隆后插入pGL3basic中的启动子片段与GenBank DNA序列数据库对比分析序列一致,且插入方向正确。在人永生化表皮细胞株(HaCat细胞)中构建的pGL3-542载体具有很强的启动活性。结论:成功构建了整合素β1远端启动子542bp片段载体,在HaCat细胞中具有非常强的启动活性。人整合素β1整合素启动子核心启动序列可能位于-845bp～-304bp。
PURPOSE: To analyze the core promoter of human β1 integrin in HaCat cells. Methods: The recombinant plasmid pGL3-1756 containing the 1745bp full-length promoter of integrin β1 gene (-1745bp ~ + 11bp) was used as a template to amplify the integrin β1 Promoter -845bp ~ -304bp gene sequence, cloned into the luciferase expression vector pGL3basic, constructed with the correct gene of interest recombinant plasmid pGL3-542. The activity of HaCat was assayed using pGL3-1756 and pGL3-542 plasmids. Results: The results of restriction enzyme digestion and sequence analysis showed that the sequence of promoter inserted into pGL3basic was the same as that of GenBank database, and the insertion direction was correct. The pGL3-542 vector constructed in human immortalized epidermal cell line (HaCat cells) has a strong priming activity. CONCLUSION: The 542bp fragment vector of integrin β1 remote promoter has been successfully constructed and has very strong promoter activity in HaCat cells. Human integrin β1 integrin promoter core promoter sequence may be located -845bp ~ -304bp.