鸡黑素皮质素受体-4反向激动剂细胞筛选模型的建立

发布时间：2018-12-23 09:29

【摘要】：本试验旨在建立体外鸡黑素皮质素受体-4(cMC4R)反向激动剂的细胞筛选模型,为从中草药中筛选出能与cMC4R作用且存在活性的中药成分提供一些基础性的研究。将成功构建的cMC4R真核表达质粒cMC4RpcDNA3.1(+)-myc与报告基因质粒pGL4.29[luc2p/CRE/Hygro]按1∶5的比例共转染到CHO-K1细胞,两周后经有限稀释法获得单克隆;将获得的单克隆分别用CRE激活剂Forskolin和MC4R激动剂NDP-MSH作用6h,挑选萤火虫荧光素酶的相对表达量最高的单克隆作为阳性细胞株;提取阳性细胞总RNA,RT-PCR检测阳性细胞内cMC4R基因、荧光素酶报告基因的转录水平;对MC4R激动剂NDP-MSH的终浓度、NDP-MSH孵育时间及溶剂DMSO的终浓度进行优化,应用Z’因子对筛选方法进行评估。结果显示,获得了5个单克隆(A2、F7、F8、F9、H10),其中单克隆A2的萤火虫荧光素酶的相对表达量最高,将其作为阳性细胞株;RT-PCR在预期位置扩增出大小分别为996和1 653bp的两条目的条带;激动剂NDP-MSH终浓度为10-9 mol/L、NDP-MSH孵育时间为8h、溶剂DMSO终浓度小于2%时,筛选模型Z’因子为0.82。结果表明,本试验所建立的筛选方法可靠、稳定,细胞筛选模型可用于cMC4R反向激动剂的筛选,为进一步从中药中筛选出有效物质奠定了基础。
[Abstract]:The aim of this study was to establish a cell screening model of chicken melanin receptor 4 (cMC4R) reverse agonist in vitro, and to provide some basic research for the screening of active components of traditional Chinese medicine which can interact with cMC4R from Chinese herbal medicine. The successfully constructed cMC4R eukaryotic expression plasmid cMC4RpcDNA3.1 ()-myc and reporter gene plasmid pGL4.29 [luc2p/CRE/Hygro] were co-transfected into CHO-K1 cells at 1:5 ratio. The obtained monoclonal cells were treated with CRE activator Forskolin and MC4R agonist NDP-MSH for 6 h, and the highest relative expression of luciferase was selected as the positive cell line. Total RNA,RT-PCR was extracted from positive cells to detect the transcription level of cMC4R gene and luciferase reporter gene. The final concentration of MC4R agonist NDP-MSH, the incubation time of NDP-MSH and the final concentration of solvent DMSO were optimized. Z' factor was used to evaluate the screening method. The results showed that five Monoclonal (A2OF7F8F8F9H10) were obtained, among which the relative expression of luciferase of Monoclonal A2 was the highest, and it was regarded as a positive cell line. The RT-PCR amplified the bands of 996 and 1 653bp items in the expected position. When the final concentration of agonist NDP-MSH was 10-9 mol/L,NDP-MSH, the incubation time was 8h, and the final concentration of solvent DMSO was less than 2, the screening model Z' factor was 0.82. The results showed that the screening method was reliable and stable, and the cell screening model could be used for the screening of cMC4R reverse agonists, which laid a foundation for the further screening of effective substances from traditional Chinese medicine.
【作者单位】：扬州大学兽医学院;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【基金】：江苏高校优势学科建设工程资助项目(PAPD) 教育部第46批“留学回国人员科研启动基金”
【分类号】：R965.1

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