以FOXP3为靶点的免疫抑制剂药物筛选模型的建立

Screening model for immuning inhibitors as target for transcription factor FOXP3

目的构建以FOXP3为靶点的免疫抑制剂筛选模型,用于筛选免疫抑制剂。方法以人类基因组DNA为模板,通过PCR扩增人类FOXP3基因启动子,将扩增的启动子DNA片段克隆至pGeneBLAzer-TOPO载体,以PCR方法确定插入片段方向,选取有正向片段的质粒克隆进行测序鉴定,用Lipofectine2000转染Jurkat细胞株,经G418抗性筛选获得阳性细胞克隆,通过PCR扩增鉴定阳性细胞克隆基因组中整合有FOXP3启动子/pGeneBLAzer-TOPO载体。以阳性对照前列腺素-2(PGE2)和PMA/Ionophore刺激细胞,探讨FOXP3启动子对下游报告子β-内酰胺酶活性的影响,并筛选了研究所内约2500余种天然产物。结果通过PCR成功扩增人类FOXP3基因启动子全长区域,构建了FOXP3启动子/pGeneBLAzer-TOPO载体,以PCR方法鉴定稳定转染细胞株Jurkat细胞基因组中整合有FOXP3启动子/pGeneBLAzer-TOPO载体,探讨了阳性对照PGE2和PMA/Ionophore对FOXP3基因启动子活性的影响,建立了以FOXP3为靶点的免疫抑制剂药物筛选模型,筛选了研究所2500余种天然产物,获得4个对FOXP3基因启动子具有上调作用的天然产物。结论建立了以FOXP3为靶点的免疫抑制剂筛选模型。

Objective Screening model for immuning inhibitors as target for transcription factor FOXP3 is established and it is used for screening immuning inhibitors.Methods Human genome was used as template and human FOXP3 gene promoter was amplified using PCR.The promoter fragment was cloned into the pGeneBLAzer-TOPO vector,the positive insert direction was selected using PCR and characterized the clone using sequence.The construct was transfected into Jurkat cell line using Lipofectine 2000 and stable transfectant was abtained using G418 selection,it was identified that the construct plasmid was integrated into stable transfectant genome using PCR.PGE2 and PMA/Ionophore were used as positive control to stimulate Jurkat cell line to assess the amount of β-lactamase reporter activity downstream of the FOXP3 gene promoter and screened about 2500 natural products.Results Full length of FOXP3 gene promoter was amplified using PCR and FOXP3 promoter/pGeneBLAzer-TOPO construct was established,it was identified that the construct plasmid was integrated into stable transfectant genome.The effective of the positive control PGE2 and PMA/Ionophore on the activity of FOXP3 gene promoter was investigated.These results indentified that the screening model is able to use for immuning inhibitors as target for transcription factor FOXP3.About 2500 natural products were screened from our institute natural products library and 4 candidate natural products which can up-regulate the activity of FOXP3 gene promoter were obtained.Conclusion Screening model for immuning inhibitors as target for transcription factor FOXP3 was established.