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Mu-opioid receptor expression in High Five insect cells is regulated by 5' untranslated region (5'UTR).

机译:高五类昆虫细胞中的阿片类鸦片受体表达受5'非翻译区(5'UTR)调控。

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摘要

There is increasing evidence that the 5'UTR of mRNAs affects regulation of gene expression in eukaryotic cells. We examined the overexpression of the mu-opioid receptor in High Five insect cells, employing rat mu-receptor cDNA linked to variable lenghts of their native 5'UTR. The sequences employed consist of either 209 nucleotides (termed ,,long") upstream the translation initiation site of the mu-receptor mRNA, or a truncated 5'UTR comprising only 11 nucleotides (,,short"). These constructs served to generate recombinant baculovirus for the expression of mu-receptor protein in High Five insect cells. 48 hours after baculovirus infection cells were harvested for mu-receptor characterization or RNA analysis. Scatchard analysis of radioligand binding consistently revealed three to four fold higher concentrations of the mu-opioid receptors expressed with the ,,long" over the ,,short" UTR containing baculovirus. The distinct expression rates of mu-receptors paralleled the amounts of mRNAs determined by RNase protection assay. Regardless of the distinct 5'UTR regions, the expressed opioid receptors displayed identical high affinity binding characteristics for the opioid antagonist diprenorphine and similar EC50 values to inhibit forskolin (10(-5) M) stimulated cAMP synthesis. Our results demonstrate that the native 5'UTR of the mu-opioid receptor has an enhancing effect on expression in the baculovirus/insect cell system.
机译:越来越多的证据表明,mRNA的5'UTR影响真核细胞中基因表达的调控。我们使用连接到其天然5'UTR可变长度的大鼠mu受体cDNA,检查了高五类昆虫细胞中mu阿片受体的过表达。所使用的序列由mu-受体mRNA翻译起始位点上游的209个核苷酸(称为“长”)或仅包含11个核苷酸的截短的5'UTR(“ short”)组成。这些构建体用于产生重组杆状病毒,用于在高五级昆虫细胞中表达mu-受体蛋白。杆状病毒感染后48小时,收获细胞用于mu受体表征或RNA分析。放射性配体结合的Scatchard分析一致地揭示了用含“长”短于“短” UTR的杆状病毒表达的μ阿片受体的浓度高了3至4倍。 mu受体的不同表达率与通过RNase保护试验确定的mRNA数量平行。不管不同的5'UTR区域如何,表达的阿片样物质受体对阿片样物质拮抗剂肾上腺素均表现出相同的高亲和力结合特性,并具有相似的EC50值来抑制毛喉素(10(-5)M)刺激的cAMP合成。我们的结果表明,μ阿片类受体的天然5'UTR对杆状病毒/昆虫细胞系统中的表达具有增强作用。

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