一种基于PXR启动子报告基因药物筛选方法的构建及其应用

doi:10.13560/j.cnki.biotech.bull.1985.2020-1440

摘要/Abstract

摘要：

构建一种基于小鼠孕烷X受体（mouse pregnane X receptor,mPXR）基因启动子双荧光素酶报告基因的药物筛选方法,并应用此方法筛选PXR的诱导剂。同源重组法设计引物,扩增mPXR基因启动子,回收片段,克隆至含有荧光素酶报告基因的pGL3-basic载体,构建pGL3-basic-PXRpro重组质粒。将构建的重组质粒和内参质粒pRL-TK共转染至Hepa1-6小鼠肝癌细胞中,给予小鼠PXR的阳性诱导剂地塞米松,24 h后检测PXR转录活性的诱导效果。利用此方法从恩诺沙星、氟苯尼考及10种连翘天然产物中筛选PXR诱导剂。经单克隆菌落PCR、重组质粒双酶切及DNA测序鉴定后获得阳性克隆,瞬时转染Hepa 1-6细胞后,通过双荧光素酶报告系统检测,所克隆的片段序列具有启动子活性,该活性可被地塞米松诱导,其相对活性为0.112 9（P<0.05）。经报告基因药物筛选方法得出,2种抗菌药物及9种连翘天然产物对PXR启动子起激活作用。成功构建了小鼠pGL3-basic-PXRpro报告基因的药物筛选方法,为筛选PXR诱导剂提供了工具。应用此报告基因法筛选了诱导PXR的天然产物,为PXR为靶点的疾病防治提供候选药物。

关键词: 孕烷X受体, 启动子, 报告基因, 药物筛选

Abstract:

The objective of this work is to construct a drug screening method based on the dual luciferase reporter gene in mouse pregnane X receptor（mPXR）gene promoter,and to apply it for screening PXR inducers. Primers were designed by homologous recombination method. The mPXR gene promoter was amplified and recovered,and cloned into the pGL3-basic vector containing the luciferase reporter gene,thus the pGL3-basic-PXRpro recombinant plasmid was constructed. Then the recombinant plasmid and the internal reference plasmid pRL-TK were co-transfected into hepa 1-6 mouse hepatocarcinoma cells. Dexamethasone,a positive inducer of PXR in mice,was administered,and the inducing effect of PXR transcription activity was tested 24 h later. Further this method was used to screen PXR inducers from enrofloxacin,florfenicol and 10 natural products of Forsythia suspensa. The positive clones were obtained after monoclonal colony PCR,recombinant plasmids double enzymatic digestion and DNA sequencing. Finally the cloned fragment sequence was detected by the dual luciferase reporter system after transiently transfecting hepa 1-6 cells. The cloned fragment sequence had promoter activity,and the activity was induced by dexamethasone,and its relative activity was 0.112 9（P<0.05）. Two antibacterial drugs and 9 natural products from F. suspense activated the PXR promoter using this reporter gene drug screening method. In conclusion,a drug screening method is successfully constructed for the mouse pGL3-basic-PXRpro reporter gene,which provides a tool for screening PXR inducers. Using this reporter gene method to screen natural products that induced PXR may provide candidate drugs for the prevention and treatment of diseases targeted by PXR.

Key words: pregnane X receptor, promoter, reporter gene, drug screening

沈雅丽, 潘阳阳, 王靖雷, 马睿, 赵改红, 王桂荣, 张倩, 王萌. 一种基于PXR启动子报告基因药物筛选方法的构建及其应用[J]. 生物技术通报, 2021, 37(9): 274-284.

SHEN Ya-li, PAN Yang-yang, WANG Jing-lei, MA Rui, ZHAO Gai-hong, WANG Gui-rong, ZHANG Qian, WANG Meng. Construction and Application of a Drug Screening Method Based on PXR Promoter Reporter Gene[J]. Biotechnology Bulletin, 2021, 37(9): 274-284.

图/表 12

表1 引物序列表

Table 1 List of primer sequence used in PCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	片段大小 Fragment length/bp
PXRpro	F：5'-gcgtgctagcccgggctcgagAGG- AACAGGTGCAGTTTG-3'	2 042
PXRpro	R：5'-cagtaccggaatgccaagcttATT- ACCTGTGTACGGCAA-3'	2 042

图1 pGL3-basic-PXRpro重组质粒构建流程

Fig.1 Construction process of pGL3-basic-PXRpro recombinant plasmid

表2 重组反应体系

Table 2 Recombination reaction system

组分 Component	重组反应 Recombination reaction/µL	阴性对照 Negative control/µL
线性化pGL3-basic载体 Linearized pGL3-basic vector	4	4
DNA片段 PXRpro DNA fragment	4	0
5× CE II Buffer	4	0
Exnase II	2	0
ddH₂O	6	16

图2 PCR扩增PXR启动子片段凝胶电泳 M1：DL 15 000 DNA marker;1：PXR启动子基因PCR扩增结果;M2：DL 2 000 Plus DNA marker

Fig.2 Gel electrophoresis of PCR amplified PXR promoter fragment M1：DL 15 000 DNA marker. 1：PXR promoter gene PCR amplification results. M2：DL 2 000 Plus DNA marker

图3 重组产物转化 A：重组反应转化板;B：阴性对照转化板

Fig.3 Recombinant product transformation A：Recombination reaction transformation plate. B：Negative control transformation plate

图4 菌落PCR凝胶电泳 M1：DL 15 000 DNA marker;1、2：构建的pGL 3-Basic-PXRpro载体单克隆菌落PCR产物;M2：DL 2 000 Plus DNA marker

Fig.4 Colony PCR gel electrophoresis M1：DL 15 000 DNA marker. 1 and 2：Constructed pGL3-Basic-PXRpro vector monoclonal colony PCR product. M2：DL 2 000 Plus DNA marker

图5 pGL3-basic-PXRpro双酶切鉴定电泳 M：DL 15 000 DNA marker;1：pGL3-basic-PXRpro双酶切产物

Fig.5 Gel electrophoresis of pGL3-basic-PXRpro by dou-ble restriction enzymes digestion M：DL 15 000 DNA marker. 1：pGL3-basic-PXRpro double restriction enzymes digestion product

图6 pGL3-basic-PXRpro的部分测序结果 A：重组质粒插入的PXR启动子片段5'端测序结果;B：重组质粒插入的PXR启动子片段3'端测序结果。红色标出的字母为突变碱基

Fig.6 Partial sequencing results of pGL3-basic-PXRpro A：Sequencing result of the 5' end of the PXR promoter fragment inserted into the recombinant plasmid. B：Sequencing result of the 3' end of the PXR promoter fragment inserted into the recombinant plasmid. The letters marked in red are mutated bases

图7 PXR启动子报告基因药物筛选方法构建的验证 A：空白组;B：空载组;C：携带报告基因载体的对照组;D：小鼠PXR的阳性诱导剂地塞米松组。图柱上标不同小写字母a、b、c和d表示组间差异显著（P<0.05）。下同

Fig.7 Validation of constructed PXR promoter reporter gene drug screening method A：Normel group. B：Empty vector group. C：Carrying reporter gene vector control group. D：A positive agonist of mPXR dexamethasone group. Different lowercase letters a,b,c,and d on the graph column indicate significant differences between groups（P<0.05）. The same below

表3 地塞米松3次独立重复实验的相对荧光素酶活性

Table 3 Relative luciferase activity of dexamethasone in three independent repeated experiments

组别 Group	N	相对荧光素酶活性 Relative luciferase activity	F	P
1	3	0.1129± 0.0100	0.200	0.824
2	3	0.1352 ± 0.0056
3	3	0.1114 ± 0.0093

图8 两种抗菌药作用Hepa 1-6细胞后的相对荧光素酶活性

Fig.8 Relative luciferase activity of two antibacterial drugs after treated in Hepa 1-6 cells

图9 10种连翘天然产物作用Hepa 1-6细胞后的相对荧光素酶活性

Fig.9 Relative luciferase activity of 10 kinds of Forsythia suspensa natural products treated on Hepa 1-6 cells

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