猪食欲肽受体2激动剂细胞筛选模型的建立及对天然中药提取物受体激动剂的筛选

doi:10.16431/j.cnki.1671-7236.2020.02.011

Abstract

Abstract: This study was aimed to construct a eukaryotic expression vector for porcine Orexin 2 receptor (pOX2R) and explore its pharmacological function when treated with traditional Chinese medicine.The pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro] were transiently transfected into HEK293T cells.The intracellular cAMP level of the positive cell lines under the action of Orexin A and Orexin B and antagonist TCS was measured by luciferase reporter gene assay.6 positive cells and 5 negative cells were obtained by stably transfecting target plasmid and reporter plasmid into CHO-K1 cells,G418 and hygromycin B were used for resistance screening.36 kinds of extracts from 18 kinds of traditional Chinese medicine were screened,HEK293T cells transfected with pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro] were used to analyze the dose effect on the positive extracts.The results showed that Orexin A and Orexin B could increase the activity of firefly luciferase,and TCS could antagonize the activation of receptor.A stable and reliable agonist screening model was constructed based on the expression level of firefly luciferase by optimizing drug stimulation time and DMSO dosage.Licorice extracted by water,lotus seed and Chinese yam extracted by alcohol could increase the expression of firefly luciferase.The extract could stimulate the expression of intracellular luciferase in a certain concentration range,which was dose-effect relationship.These results would provide reference for screening compounds and related drugs that could improve pig production efficiency.

Key words: pig; Orexin-2 receptor(pOX2R); firefly luciferase; traditional Chinese medicine; screening

CLC Number:

Q782

CUI Zihe, ZHANG Haijie, LIN Shuai, JIANG Junna, XIAO Xia, LIU Yuan, WANG Zhiqiang. A Establishment of Agonist Screening Model Based on Pig Orexin-2 Receptor and Screening of Receptor Agonists for Traditional Chinese Medicine[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 407-415.

References

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A Establishment of Agonist Screening Model Based on Pig Orexin-2 Receptor and Screening of Receptor Agonists for Traditional Chinese Medicine

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