pRluc-hNeurotensinl-R重组真核表达载体的构建及在离体细胞中的表达  

Construction of pRIuc-hNTSR1 -pcDNA3.1 eukaryotic expression vectors and its expression in isolated cells

作　　者：蔡欣[1] 陈京[1] 白波[1] 

机构地区：[1]济宁医学院神经生物研究所,济宁272067

出　　处：《中华行为医学与脑科学杂志》2011年第12期1140-1142,共3页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：国家自然科学基金资助项目（30971081;30870932;81070961）;山东省自然科学基金资助项目（Y2007D01;ZR2009DZ004）

摘　　要：目的构建与海肾荧光素酶（Rluc）融合的人Neurotensin-R1（HumanNeurotensinreceptorl，NTS1 or NTSR1）真核表达载体，用于生物发光共振能量转移法检测人NTS与其它受体间的相互作用以及研究Neurotensin—R介导的细胞内信号转导机制。方法以质粒peDNA3．1-hNTS1为模板，PCR方法扩增人NTS。扩增的人NTS以及质粒pRluc—pcDNA3．1用NotI和XbaI双酶切，然后将这2种酶切产物按常规方法连接、转化至大肠杆菌Top10中，该菌在培养箱中孵育12～16h后，挑取菌落培养，提取质粒，进行酶切鉴定，最后进行测序。将测序正确的重组载体用脂质体法转染人胚胎肾（humanembryonickidney293，HEK293）细胞。最后，通过共聚焦显微镜观察经过免疫荧光染色的细胞以及Westernblot鉴定人Neu—rotensinl—R的表达。结果通过PCR扩增出一条1257bp的基因片段，序列与GenBank（NM-002531）相同。Westernblot中大约90kDa处有一蛋白条带，与预期大小相同。人Neurotensin—R表达在细胞膜上。结论成功构建了pRluc—hNeurotensinl—R重组表达载体，建立了该质粒转染HEK293的细胞模型。可被用于检测与其它受体间的相互作用以及研究Neurotensinl—R介导的细胞内信号转导机制，这将有助于探究疾病的发病机制及开发新的药物靶点。Objective To construct expression vectors that Renilla reuiformis （ Rluc ） fused with neuro- tensin 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 peDNA3.1-hNTSR1 as template. The PCR product was digested,ligased with the plasmid pRlue and then be transformed into the competent cell Topl0. The construct was identified by DNA sequencing. The recombinant plasmid was transiently transfeeted into human embryonic kidney 293 （HEK293） cells, and the expression of pRluc-hNTSRI-pcDNA3.1 was detected by confocal microscopy and Western blot. Re- suits 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-hNTSRI-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 pro- vide new target for drug development.

关 键 词：Neurotensin—R1 海肾荧光素酶 真核表达载体 HEK293细胞 

分 类 号：Q78[生物学—分子生物学]