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采用PCR方法扩增了ZNF313基因的启动子序列,构建了含人ZNF313基因启动子不同片断的荧光素酶报告基因表达体系.以pRLTK为内参照质粒,瞬时转染HEK293T细胞,48h后收集细胞,测定荧光素酶的相对表达活性.结果发现,在ZNF313基因的启动子区域构建了4种荧光素酶报告基因表达体系,即pGL3215(-215bp～+38bp)、pGL3160(-160bp～+38bp)、pGL3133(-133bp～+128bp)和pGL38(-8bp～+128bp).其中pGL3215表达载体的荧光素酶相对表达活性最高;pGL3160和pGL3133表达载体的荧光素酶相对表达活性几乎相同,且是pGL3215的75%;而pGL38的荧光素酶相对表达活性急剧下降,接近于零.这表明,-133bp～-8bp区域内含有人ZNF313基因转录所必需的启动子序列.生物信息学的分析表明,两个SP1、一个AP2和一个TAg是人ZNF313基因启动子所必需的.图4参19
The promoter sequence of ZNF313 gene was amplified by PCR and the luciferase reporter gene expression system containing different fragments of human ZNF313 promoter was constructed.Transfection of HEK293T cells was performed with pRLTK as an internal reference and the cells were harvested after 48 hours, The results showed that four luciferase reporter gene expression systems were constructed, namely pGL3215 (-215bp ~ + 38bp), pGL3160 (-160bp ~ + 38bp) pGL3133 (-133bp ~ + 128bp) and pGL38 (-8bp ~ + 128bp), of which pGL3215 expression vector had the highest relative luciferase activity and luciferase relative expression activities of pGL3160 and pGL3133 expression vector were almost the same, 75%, while the relative expression of pGL38 luciferase activity decreased sharply, close to zero.This shows that -133bp ~ -8bp region contains the human ZNF313 gene transcription necessary promoter sequence analysis of bioinformatics showed that two SP1, one AP2 and one TAg are required for the human ZNF313 gene promoter.