[1]曾凡新,董志,傅洁民,等.人β3肾上腺素受体真核表达质粒pcDNA3.1(+)-β3-AR的构建及表达[J].第三军医大学学报,2007,29(21):2070-2072.
 ZENG Fan-xin,DONG Zhi,FU Jie-min,et al.Construction and expression of a eukaryotic β3-adrenoceptor expression vector pcDNA3.1(+)-β3-AR[J].J Third Mil Med Univ,2007,29(21):2070-2072.
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人β3肾上腺素受体真核表达质粒pcDNA3.1(+)-β3-AR的构建及表达(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第21期
页码:
2070-2072
栏目:
论著
出版日期:
2007-11-15

文章信息/Info

Title:
Construction and expression of a eukaryotic β3-adrenoceptor expression vector pcDNA3.1(+)-β3-AR
作者:
曾凡新董志傅洁民刘晓海
重庆医科大学药学院药理学教研室;重庆医药工业研究院药理毒理中心
Author(s):
ZENG Fan-xin DONG Zhi FU Jie-min LIU Xiao-hai
Department of Pharmacology, Chongqing Medical University, Chongqing 400016, Center of Pharmacology and Toxicology, Chongqing Pharmaceutical Research Institute, Chongqing 400061, China
关键词:
β3肾上腺素受体真核表达载体HEK293细胞
Keywords:
β3-adrenoceptor eukaryotic expression vector HEK293 cell
分类号:
R394-33; R394.3; R965.2
文献标志码:
A
摘要:
目的  构建人β3肾上腺素受体 (β3-AR)的真核表达质粒pcDNA3.1(+)-β3-AR,并通过脂质体介导转染HEK293细胞使之表达该受体蛋白。  方法  以pENTR_ADRB3-Stop质粒为模板,采用PCR法扩增出人β3-AR cDNA,将其插入真核表达载体pcDNA3.1(+)中,构建成真核表达质粒pcDNA3.1(+)-β3-AR。将该质粒转染HEK293真核细胞,通过RT-PCR检测β3-AR mRNA在HEK293细胞内的表达,通过免疫荧光法检测β3-AR蛋白在HEK293细胞中的表达。  结果  酶切和DNA测序鉴定均证实重组质粒含有人β3-AR编码区全长。在转染重组质粒pcDNA3.1(+)-β3-AR的HEK293细胞中检测到了β3-AR基因的转录和翻译产物。  结论  通过基因工程方法成功构建了β3-AR的真核表达质粒pcDNA3.1(+)-β3-AR,为进一步建立β3-AR的稳定表达细胞株,用于筛选高效、高选择性β3-AR激动剂奠定了基础。
Abstract:
Objective    To construct the recombinant eukaryotic expression vector pcDNA3.1(+)-β3-AR and express it in HEK293 cells.    Methods    Human β3-AR cDNA was amplified by PCR from a pENTR_ADRB3-Stop plasmid, and the eukaryotic expression plasmid pcDNA3.1(+)-β3-AR was constructed by inserting the β3-AR cDNA into KpnⅠ/XbaⅠ-digested pcDNA3.1(+). The DNA sequence was confirmed by double digestion, and the pcDNA3.1(+)-β3-AR plasmid was transfected into HEK293 cell line. Human β3-AR mRNA expression on HEK293 cells was detected by RT-PCR, and the protein expression on HEK293 cells was detected by immunofluorescence technique.    Results    Restriction analysis and DNA sequencing showed that the recombinant plasmid contained the whole coding region of human β3-AR gene. RT-PCR assay indicated the transcription of β3-AR mRNA and the β3-AR protein detectable in the transfected HEK293 cells.     Conclusion    Recombinant eukaryotic expression vector pcDNA3.1(+)-β3-AR plasmid was successfully constructed by genetic engineering technique and expressed on the surface of HEK293 cells, which will contribute to further studies on the establishment of stable expression cell lines for screening high potent and selective β3-AR agonists.

参考文献/References:

曾凡新,董志,傅洁民,等.  人β3肾上腺素受体真核表达质粒pcDNA3.1(+)-β3-AR的构建及表达[J]. 第三军医大学学报, 2007, 29(21):2070-2072.

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更新日期/Last Update: 2008-07-02