猪食欲肽2受体突变体真核表达载体的构建及其对cAMP信号通路的影响

doi:10.16431/j.cnki.1671-7236.2018.07.005

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (7): 1768-1774.doi: 10.16431/j.cnki.1671-7236.2018.07.005

猪食欲肽2受体突变体真核表达载体的构建及其对cAMP信号通路的影响

刘敏^1,2, 闵天奇^1,2, 张海洁^1,2, 谢华杰^1,2, 崔子鹤^1,2, 王志强^1,2

1. 扬州大学兽医学院, 兽医药理毒理教研室, 扬州 225009;
2. 江苏高校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:2018-01-25 出版日期:2018-07-20 发布日期:2018-07-20
通讯作者: 王志强 E-mail:zqwang@yzu.edu.cn
作者简介:刘敏(1994-),女,江苏淮安人,硕士生,研究方向:兽医药理学与毒理学,E-mail:977034892@qq.com
基金资助:
国家自然科学基金项目（31572571）；江苏高校优势学科建设工程资助项目（PAPD）

Construction of the Eukaryotic Expression Plasmid for Mutants of Porcine Orexin 2 Receptor and Their Effect on cAMP Signaling Pathways

LIU Min^1,2, MIN Tianqi^1,2, ZHANG Haijie^1,2, XIE Huajie^1,2, CUI Zihe^1,2, WANG Zhiqiang^1,2

1. Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2018-01-25 Online:2018-07-20 Published:2018-07-20

摘要/Abstract

摘要：

试验旨在构建猪食欲肽2受体（porcine orexin 2 receptor，pOX2R）突变体的真核表达载体，探究其野生型与突变体的基础药理学活性差异。以pcDNA3.1（+）-myc/pOX2R野生型质粒为模板，设计特异性引物单点突变构建4种突变体：pcDNA3.1（+）-myc/pOX2R-P10S、pcDNA3.1（+）-myc/pOX2R-P11T、pcDNA3.1（+）-myc/pOX2R-V308I和pcDNA3.1（+）-myc/pOX2R-T401I，将pcDNA3.1（+）-myc/pOX2R野生型和4种突变体分别瞬时转染HEK293T细胞，利用双荧光素酶报告基因检测法测定pOX2R野生型及突变体的基础活性，并检测不同浓度激动剂作用下细胞内cAMP水平，随后用内源性激动剂食欲肽A （OXA）及食欲肽B （OXB）分别对野生型及突变体进行刺激。结果显示，4个突变体构建成功，pOX2R的第10、11、308和401位氨基酸分别突变为丝氨酸、苏氨酸、异亮氨酸和缬氨酸。将pOX2R的野生型及4个突变体瞬时转染HEK293T细胞后，野生型与突变体的基础活性值无显著差异（P>0.05），表明这4个位点的氨基酸突变对其受体的基础表达信号无显著影响。与野生型受体相比，突变型受体对激动剂OXB的响应无显著差异（P>0.05），而突变体P10S、P11T和T401I对激动剂OXA的响应EC₅₀显著降低（P<0.05），其R_max无显著差异（P>0.05）。推测第10、11和401位点的氨基酸突变可能影响了激动剂OXA与受体的结合，降低了激动剂的激动效应。本研究结果为进一步体外研究pOX2R的功能奠定了基础。

关键词: 猪; 食欲肽2受体(OX2R)基因; 突变体; cAMP

Abstract:

This study were aimed to construct the eukaryotic expression vectors for porcine orexin 2 receptor (pOX2R) mutants and explore the difference in constitutive activity between wild type and mutants.We used the eukaryotic expression vector pcDNA3.1(+)/pOX2R-myc WT as the template to construct four mutants,named pcDNA3.1(+)-myc/pOX2R-P10S,pcDNA3.1(+)-myc/pOX2R-P11T,pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I,and then transient transfected the plasmids into HEK293T cell.The basal activities of pOX2R wild-type and mutants were determined using dual luciferase reporter assays,the intracellular cAMP levels were measured at different concentrations of agonists,the wild-type and mutants were stimulated with the endogenous agonists orexin A (OXA) and orexin B (OXB),respectively.The results showed that the recombinant plasmid pcDNA3.1(+)/pOX2R-myc-P10S,pcDNA3.1(+)-myc/pOX2R-P11T,pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I were constructed successfully.The 10,11,308 and 401 amino acids of the pOX2R were successfully mutated to serine,threonine,isoleucine and valine,respectively.There were no significant difference between reconstruction plasmid of pcDNA3.1(+)-myc/pOX2R WT and four mutants in basal activity (P>0.05).It showed that there was no significant effect of amino acid mutations at 4 sites on the basal expression signal of their receptors.Compared with the wild-type receptor,there was no significant difference in the response of the mutant receptor to OXB (P>0.05),but the EC₅₀ of the mutant P10S,P11T and T401I to agonist OXA was significantly lower (P<0.05),there was no significant difference in R_max (P>0.05).The mutants (P10S,P11T and T401I) might be affect the binding between agonist OXA ligand and receptor,and reduce the agonistic effect of the agonist.The study provided foundation for the further exploration of the function of pOX2R.The results laid the foundation for further study of pOX2R function in vitro.

Key words: porcine; OX2R gene; mutant; cAMP

中图分类号:

S828

刘敏, 闵天奇, 张海洁, 谢华杰, 崔子鹤, 王志强. 猪食欲肽2受体突变体真核表达载体的构建及其对cAMP信号通路的影响[J]. 《中国畜牧兽医》, 2018, 45(7): 1768-1774.

LIU Min, MIN Tianqi, ZHANG Haijie, XIE Huajie, CUI Zihe, WANG Zhiqiang. Construction of the Eukaryotic Expression Plasmid for Mutants of Porcine Orexin 2 Receptor and Their Effect on cAMP Signaling Pathways[J]. , 2018, 45(7): 1768-1774.

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猪食欲肽2受体突变体真核表达载体的构建及其对cAMP信号通路的影响

Construction of the Eukaryotic Expression Plasmid for Mutants of Porcine Orexin 2 Receptor and Their Effect on cAMP Signaling Pathways

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