Construction and Application of a Drug Screening Method Based on PXR Promoter Reporter Gene

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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

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

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

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