生物技术通报 ›› 2021, Vol. 37 ›› Issue (9): 274-284.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1440
沈雅丽(), 潘阳阳, 王靖雷, 马睿, 赵改红, 王桂荣, 张倩, 王萌()
收稿日期:
2020-11-18
出版日期:
2021-09-26
发布日期:
2021-10-25
作者简介:
沈雅丽,女,硕士研究生,研究方向:兽医药理学与毒理学基础;E-mail: 基金资助:
SHEN Ya-li(), PAN Yang-yang, WANG Jing-lei, MA Rui, ZHAO Gai-hong, WANG Gui-rong, ZHANG Qian, WANG Meng()
Received:
2020-11-18
Published:
2021-09-26
Online:
2021-10-25
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。
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.
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 |
R:5'-cagtaccggaatgccaagcttATT- ACCTGTGTACGGCAA-3' |
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 |
R:5'-cagtaccggaatgccaagcttATT- ACCTGTGTACGGCAA-3' |
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 |
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
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 |
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 |
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