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目的 :构建大鼠补体应答基因32(response gene to complement,RGC32)启动子(全长和截短)荧光素酶报告质粒,并观察人胚肾细胞(HEK293)过表达髓锌指基因1(myeloid zinc finger gene1,MZF1)对大鼠RGC32基因启动活性的影响。同时,筛选其可能的MZF1结合位点。方法:将RGC32基因启动子全长(-686~-1 nt)插入到荧光素酶报告基因载体p GL3-basic中,获得RGC32基因启动子全长荧光素酶报告质粒(p GL3-RGC32-FL)后,再将p GL3-RGC32-FL与本课题组前期构建的大鼠野生型MZF1表达质粒(p IRES2-EGFP-MZF1)共转染HEK293细胞,检测其荧光素酶活性,以确定MZF1对RGC32基因的启动作用。另用生物信息学软件预测RGC32基因启动子上转录因子MZF1潜在的结合位点,并据此构建3个RGC32基因启动子截短的荧光素酶报告质粒(即p GL3-RGC32-1、p GL3-RGC32-2和p GL3-RGC32-3)。将上述RGC32基因启动子全长和各截短的荧光素酶报告质粒与MZF1过表达质粒共转染HEK293细胞,再行荧光素酶活性测定,以筛选MZF1的结合位点。结果:菌液PCR及核酸测序证实,大鼠RGC32基因启动子(全长和各截短)的荧光素酶报告质粒均构建成功。将p GL3-RGC32-FL和p IRES2-EGFPMZF1共转染HEK293细胞发现,RGC32基因启动子活性显著增加。而将p GL3-RGC32-FL、p GL3-RGC32-1、p GL3-RGC32-2和p GL3-RGC32-3分别与p IRES2-EGFP-MZF1共转染HEK293细胞后显示,p GL3-RGC32-3的启动活性显著低于p GL3-RGC32-FL、p GL3-RGC32-1和p GL3-RGC32-2。提示MZF1可能结合在RGC32基因启动子的-286~-86 nt区域。结论 :成功构建了大鼠RGC32基因启动子全长及截短荧光素酶报告质粒,证实过表达MZF1可促进RGC32基因的启动,并初步筛查出转录因子MZF1在RGC32基因启动子上可能的结合区域。
OBJECTIVE: To construct luciferase reporter plasmids of rat gene complement (RGC32) 32 (full length and truncated), and observe the effect of myeloid overexpression on human embryonic kidney cells (HEK293) zinc finger gene1, MZF1) on the activation of RGC32 gene in rats. At the same time, the possible MZF1 binding sites were screened. Methods: The full-length of RGC32 gene promoter (-686 ~ -1 nt) was inserted into luciferase reporter plasmid pGL3-basic to obtain full-length luciferase reporter plasmid of RGC32 promoter (pGL3-RGC32-FL ), PGL3-RGC32-FL was co-transfected into wild type MZF1 expression plasmid (pIRES2-EGFP-MZF1) co-transfected with pGL3-RGC32-FL into HEK293 cells to detect the luciferase activity. RGC32 gene promoter. Bioinformatics software was also used to predict the potential binding site of transcription factor MZF1 on the promoter of RGC32 gene and to construct three luciferase reporter plasmids (ie, pGL3-RGC32-1, pGL3 -RGC32-2 and pGL3-RGC32-3). The full length of the RGC32 promoter and the truncated luciferase reporter plasmid and the MZF1 overexpression plasmid were co-transfected into HEK293 cells and luciferase activity assay was performed to screen the binding sites of MZF1. Results: Bacterial PCR and DNA sequencing confirmed that the luciferase reporter plasmid of rat RGC32 promoter (full length and truncated) was successfully constructed. Co-transfection of pGL3-RGC32-FL and pIRES2-EGFPMZF1 into HEK293 cells showed that the promoter activity of RGC32 gene was significantly increased. However, when pGL3-RGC32-FL, pGL3-RGC32-1, pGL3-RGC32-2 and pGL3-RGC32-3 were cotransfected into HEK293 cells with pIRES2-EGFP-MZF1 respectively, pGL3-RGC32- 3 was significantly lower than pGL3-RGC32-FL, pGL3-RGC32-1 and pGL3-RGC32-2. Suggesting that MZF1 may bind to the -286 ~ -86 nt region of the RGC32 gene promoter. CONCLUSION: The full-length and truncated luciferase reporter plasmid of rat RGC32 gene was constructed successfully. It was confirmed that MZF1 overexpression could promote the initiation of RGC32 gene and preliminarily screen the possible binding of transcription factor MZF1 on the promoter of RGC32 gene area.