Construction of pRluc-hNTSR1-pcDNA3.1 eukaryotic expression vectors and its expression in isolated cells
10.3760/cma.j.issn.1674-6554.2011.12.028
- VernacularTitle:pRluc-hNeurotensin1-R重组真核表达载体的构建及在离体细胞中的表达
- Author:
Cai XIN
;
Chen JING
;
Bai BO
- Publication Type:Journal Article
- Keywords:
Neurotensin type 1 receptor;
Renilla reniformis;
Eukaryotic expression vector;
HEK293 cells
- From:
Chinese Journal of Behavioral Medicine and Brain Science
2011;20(12):1140-1142
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct expression vectors that Renilla reniformis (Rluc) fused with neurotensin type 1 receptor (NTSR1),and to investigate the interaction between NTSR1 and other receptors,as well as intercellular signal transduction mechanism mediated by neurotensinl-R.Methods The human NTSR1 gene was amplified by PCR using the plasmid pcDNA3.1-hNTSR1 as template.The PCR product was digested,ligased with the plasmid pRluc and then be transformed into the competent cell Top10.The construct was identified by DNA sequencing.The recombinant plasmid was transiently transfected into human embryonic kidney 293 ( HEK293 )cells,and the expression of pRluc-hNTSR1-pcDNA3.1 was detected by confocal microscopy and Western blot.Results The fragment of 1257 bp was amplified by PCR,and the DNA sequences were identical with the gene in GenBank ( NM_002531 ).Western blot showed a band about 90kDa.Confocal microscopy showed that NTSR1 was expressed on the plasma membrane.Conclusion The pRluc-hNTSR1-pcDNA3.1 eukaryotic expression vector is successfully constructed,and the expression vector can be used to investigate the interaction between NTSR1 and other receptors,as well as intercellular signal transduction mechanism mediated by neurotensinl-R,which will provide new target for drug development.
