鸡黑素皮质素受体-4反向激动剂细胞筛选模型的建立

doi:10.16431/j.cnki.1671-7236.2015.07.006

›› 2015, Vol. 42 ›› Issue (7): 1661-1667.doi: 10.16431/j.cnki.1671-7236.2015.07.006

鸡黑素皮质素受体-4反向激动剂细胞筛选模型的建立

周家华^1,2, 李栋^1,2, 姜军娜^1,2, 柴继田^1,2, 王建兵^1,2, 李颖颖^1,2, 王志强^1,2

1. 扬州大学兽医学院, 扬州 225009;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2015-01-26 出版日期:2015-07-20 发布日期:2015-07-23
通讯作者: 王志强 E-mail:zqwang@yzu.edu.cn
作者简介:周家华(1990-),男,江西瑞金人,硕士生,研究方向:兽医药理与毒理学,E-mail:13665243517@163.com
基金资助:
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"

Establishment of Cell Screening Model for Discovering Antagonists of Chicken Melanocortin-4 Receptor

ZHOU Jia-hua^1,2, LI Dong^1,2, JIANG Jun-na^1,2, CHAI Ji-tian^1,2, WANG Jian-bing^1,2, LI Ying-ying^1,2, WANG Zhi-qiang^1,2

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Co-innovation Center for Prevention and Control of Important Animal Infections Disease and Zoonoses, Yangzhou 225009, China

Received:2015-01-26 Online:2015-07-20 Published:2015-07-23

摘要/Abstract

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cMC4R-pcDNA3.1(+)-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6 h,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1 653 bp的两条目的条带;激动剂NDP-MSH终浓度为10^-9mol/L、NDP-MSH孵育时间为8 h、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。

关键词: 鸡黑素皮质素受体-4(cMC4R); 报告基因; 细胞筛选模型

Abstract: This test was intended to establish a cell screening model for discovering new antagonists of chicken melanocortin-4 receptor (cMC4R) in vitro,and provide some basic research for screening ingredients which could interact with cMC4R and have activities from traditional Chinese medicine.The eukaryotic expression plasmid cMC4R-pcDNA3.1(+)-myc and a reporter gene plasmid pGL4.29[luc2p/CRE/Hygro] were co-transfected with the ratio of 1:5 into CHO-K1 cell line.Monoclone were obtained by limiting dilution method two weeks later.The obtained monoclone reacted with Forskolin and NDP-MSH for 6 h,respectively,choosing the monoclonal cell line which the relative expression of firefly luciferase was highest as a positive.Extract total RNA of positive cells,and detect the transcription of cMC4R gene and luciferase reporter gene by RT-PCR.To identify and optimize the assay condition,the effects of some factors were examined using NDP-MSH,including final concentration of NDP-MSH,incubation time,and final concentration of DMSO,and using Z'-factor to evaluate the screening method.The results showed that 5 monoclone (A2,F7,F8,F9 and H10)were obtained,while took A2 as the positive cell lines in which the relative expression of firefly luciferase was highest;The cMC4R gene (996 bp),luciferase reporter gene (1 653 bp) were detected in the A2 cell lines by RT-PCR;A stable cell line was established for cMC4R antagonist screening,the Z'-factor was 0.82 on the condition the final concentration of NDP-MSH was 10^-9 mol/L,the incubation time was 8 h and the final concentration of DMSO was less than 2%.The results showed that the established cell screening model could be used in the screening of cMC4R antagonists,and laid the foundation for extracting the effective substances from traditional Chinese medicine.

Key words: chicken melanocortin-4 receptor (cMC4R); reporter gene; cell screening model

中图分类号:

R965.1

周家华, 李栋, 姜军娜, 柴继田, 王建兵, 李颖颖, 王志强. 鸡黑素皮质素受体-4反向激动剂细胞筛选模型的建立[J]. , 2015, 42(7): 1661-1667.

ZHOU Jia-hua, LI Dong, JIANG Jun-na, CHAI Ji-tian, WANG Jian-bing, LI Ying-ying, WANG Zhi-qiang. Establishment of Cell Screening Model for Discovering Antagonists of Chicken Melanocortin-4 Receptor[J]. , 2015, 42(7): 1661-1667.

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鸡黑素皮质素受体-4反向激动剂细胞筛选模型的建立

Establishment of Cell Screening Model for Discovering Antagonists of Chicken Melanocortin-4 Receptor

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