大鼠delta阿片受体短发卡RNA干扰真核表达载体的构建和鉴定

Construction and identification of delta opioid receptor-short hairpin RNA eukaryotic expression vector

背景:长期应用阿片受体治疗疼痛会导致药物耐受或成瘾,可能与delta阿片受体密度上调有关。目的:设计及构建大鼠delta阿片受体短发卡RNA真核表达载体并鉴定。方法:根据大鼠delta阿片受体mRNA序列设计并体外合成短发卡RNA寡核苷酸片段,退火形成双链,克隆到线性化质粒pGenesil-1,然后进行酶切和测序鉴定。结果与结论:酶切证明delta阿片受体-短发卡RNA已经插入到质粒载体pGenesil-1里,测序结果证明均为插入正确的克隆质粒,而且质量均符合设计标准,证实靶向大鼠delta阿片受体基因的短发卡RNA真核表达载体构建成功。

BACKGROUND：Long-term application of opioid receptor for treatment of pains would cause drug tolerance or addiction,which is possibly related to upregulated delta opioid receptor（DOR） density.OBJECTIVE：To design and construct short hairpin RNA（shRNA） eukaryotic expression plasmids targeting DOR gene which may play an important role in morphine tolerance.METHODS：Three pairs of short chain oligonucleotides targeted to rat DOR mRNA（Accession No：NM_012617） were designed and synthesized individually based on the sequence of rat DOR mRNA at first.Then the synthestic sense and antisense oligonucleotide strands were mixed together in annealing buffer to form 3 DNA duplexes.Subsequently,the DNA segments were cloned into pGenesil-1.2 vectors respectively.At last,the plasmids were identified by restriction analysis and sequencing test.RESULTS AND CONCLUSION：The results of restriction analysis and sequencing test showed that all three designed DOR shRNA-expression duplexes were successfully inserted into the plasmid vector pGenesil-1.2 respectively and recombinant plasmid vectors were constructed meeting our aims.The DOR-shRNA expression vectors were constructed successfully and could be used in RNAi＇s study on morphine tolerance.