胆汁酸膜受体TGR5真核表达载体的构建及在细胞中的表达

中国畜牧兽医

胆汁酸膜受体TGR5真核表达载体的构建及在细胞中的表达

史琳凯¹^，2，刘倩^1，2，张子英^1，2，刘畅¹，王旭东¹^，2，丁月霞^1，2，李培锋^1，2

（1.内蒙古农业大学兽医学院，内蒙古呼和浩特 010018；2.农业部动物疾病临床诊疗技术重点实验室，内蒙古呼和浩特 010018）

收稿日期:2013-12-16 出版日期:2014-05-20 发布日期:2014-06-25
通讯作者: 李培锋。E-mail: lpfneimeng@163.com
作者简介:史琳凯（1988—），女，内蒙古人，硕士生，研究方向：兽医药理学与毒理学。
基金资助:
国家自然科学基金(31272605)。

Construction of Bile Acid Receptor TGR5 Eukaryotic Expression Vector and its Expression in Cell

SHI Lin-kai¹^，2, LIU Qian¹^，2, ZHANG Zi-ying¹^，2, LIU Chang¹， WANG Xu-dong¹^，2, DING Yue-xia¹^，2, LI Pei-feng¹^，2

(1. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; 2. Key Laboratory of Clinical Diagnosis and Treatment Techniques in Animal Disease, Ministry of Agriculture, Hohhot 010018, China)

Received:2013-12-16 Online:2014-05-20 Published:2014-06-25

摘要/Abstract

摘要： 试验旨在构建胆汁酸膜受体TGR5真核表达载体，转染293T细胞并在其中表达。用RT-PCR技术从胎盘组织中得到TGR5基因，克隆至真核表达载体pCMV-EGFP中。双酶切和测序鉴定正确后，将pCMV-EGFP-TGR5瞬时转染293T细胞，并采用实时荧光定量PCR和Western blotting技术检测TGR5的表达。结果显示，本试验构建了TGR5真核表达载体pCMV-EGFP-TGR5；转染293T细胞后，荧光显微镜观察到绿色荧光表达；实时荧光定量PCR检测TGR5表达量显著增加；Western blotting结果显示有目的蛋白表达。结果表明，真核表达载体pCMV-EGFP-TGR5构建成功，转染293T细胞后在基因、蛋白水平上均表达TGR5受体。

关键词: font-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA" lang="EN-US">TGRfont-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA" lang="EN-US">5font-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">基因；克隆；真核表达载体构建

Abstract: The study was aimed to construct a membrane-bound bile acid receptor TGR5 eukaryotic expression vector, which transfected into 293T cell, and expressed in 293T cell. Total RNA was extracted from placenta tissue, and RT-PCR was performed to obtain the TGR5 cDNA, which was inserted into the eukaryotic expression vector pCMV-EGFP. The novel constructed plasmid was confirmed by restriction enzyme digestion and DNA sequencing, then 293T cell was transfected with pCMV-EGFP-TGR5, Real-time PCR and Western blotting were amplified to determine the expression of TGR5. The result showed that the eukaryotic expression vector of pCMV-EGFP-TGR5 was constructed successfully. Green fluorescent was examined under fluorescent microscope, and the expression of TGR5 was increased significantly in 293T cells. The eukaryotic expression vector pCMV-EGFP-TGR5 was constructed successfully and TGR5 was expressed in mRNA and protein levels in 293T cell after transfection.

Key words: font-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA" lang="EN-US">TGRfont-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA" lang="EN-US">5 gene; clone; construction of eukaryotic expression vector

史琳凯，刘倩，张子英，刘畅，王旭东，丁月霞，李培锋. 胆汁酸膜受体TGR5真核表达载体的构建及在细胞中的表达[J]. 中国畜牧兽医.

SHI Lin-kai, LIU Qian, ZHANG Zi-ying, LIU Chang， WANG Xu-dong, DING Yue-xia, LI Pei-feng. Construction of Bile Acid Receptor TGR5 Eukaryotic Expression Vector and its Expression in Cell[J]. .

参考文献

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