pRluc-KOR真核表达载体的构建及在HEK293细胞中的表达
Construction of pRluc-KOR eukaryotic expression vector and its expression in HEK293 cells
李雅林 1白波 2陈京 3刘有旺 3刘海青 3于鹏丽3
作者信息
- 1. 泰山医学院基础部神经生物学实验室,山东,泰安,271000;山东农业大学生命科学学院,山东,泰安,271018
- 2. 济宁医学院神经生物学实验室,山东,济宁,272000
- 3. 泰山医学院基础部神经生物学实验室,山东,泰安,271000
- 折叠
摘要
目的:构建与海肾萤光素酶(Rluc)融合的K型阿片受体(KOR)真核表达载体,用于生物发光共振能量转移法(BRET)检测apelin受体和KOR之间的相互作用. 方法:以质粒peDNA3.1-KOR为模板,PCR方法扩增KOR.扩增的KOR用Not Ⅰ和Xho Ⅰ双酶切,同时用这两种酶双酶切质粒pRluc.将这两种酶切产物按常规方法连接、转化大肠杆菌Top10.挑取菌落培养,提取质粒,行酶切鉴定及测序.将测序正确的重组载体用脂质体法转染人胚胎肾(HEK293)细胞,免疫荧光染色,共聚焦显微镜观察. 结果:扩增出了1条1143 bp的片段,与预期的KOR大小相符,质粒酶切结果显示pRluc-KOR被切成2条片段,其中一条为pRluc载体大小,另一条为目的片段大小.经测序鉴定,序列与GenBank(NM_000912)中的序列高度同源.共聚焦显微镜观察显示,阿片受体主要表达在细胞膜上. 结论:构建成功了pRluc-KOR重组表达载体,此表达载体可用于检测KOR与apelin受体或与其他受体之间的相瓦作用.
Abstract
AIM: To construct eukaryotic expression vector pRluc-KOR containing Renilla luciferase gene and kappa opioid receptor and to detect interactions between apelin receptor and kappa opioid receptor by bioluminescence resonance energy transfer (BRET). METHODS: Human kappa opioid receptor(KOR) was amplified by PCR using plasmid pcDNA 3.1- KOR as template. The PCR product was purified by agarose gel extraction and digested with Not Ⅰ and Xho Ⅰ, and then was inserted between the Not Ⅰ and Xho Ⅰ, sites of pRluc (N-terminal) (Hind Ⅲ/EcoRV) upstream from the 5'-end of Rluc cDNA. The construct was identified by sequencing. The recombinant plasmid was transiently transfected into human embryonic kidney 293 (HEK293) cells and the expression of pRluc-KOR was observed by confocal microscopy. RESULTS; A fragment of 1143 bp was amplified by PCR, which was of the same size as the anticipated kappa opioid receptor. The recombinant plasmid was digested into two fragments, one was 1143 bp and the other was pRluc vector, and the sequence of the KOR was identical with the gene in the Gene Bank (NM_000912). Confocal microscopy showed that KOR was expressed on the plasma membrane. CONCLUSION: The pRluc-KOR eukaryotic expression vector is successfully constructed and the expression vector can be used to detect interactions between KOR and other receptors.
关键词
K型阿片受体/真核表达载体/构建/生物发光共振能量转移Key words
kappa opioid receptors/eukaryotic expression vector construction/bioluminescence resonance energy transfer引用本文复制引用
基金项目
国家自然科学基金(30870932)
山东省自然科学基金(Y2005CA7)
山东省科技攻关计划(2006GG2202037)
山东省自然科学基金(Y2007D01)
出版年
2009
NETL
NSTL
维普
万方数据