大鼠delta阿片受体shRNA干扰真核表达载体的构建和鉴定.docVIP

  大鼠 delta 阿片受体 shRNA 干扰真核表达 载体的构建和鉴定# 胡松权，杨辉，王鹏** 5 10 15 20 25 30 35 40 （华中科技大学同济医学院附属同济医院麻醉科，武汉 430030） 摘要：目的 设计、构建大鼠 delta 阿片受体（delta opioid receptor, DOR）shRNA 真核 表达载体并鉴定。方法 根据大鼠 DOR mRNA 序列设计并体外合成 shRNA 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 酶切证明 DOR-shRNA 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。 结论 靶向大鼠 DOR 基因的 shRNA 真核表达载体构建成功，为 质粒的筛选和进一步研究 DOR 在吗啡耐受中的作用与机制奠定了基础。 关键词：DOR；RNA 干扰；吗啡耐受；载体构建 中图分类号：R614 Construction and identification of DOR-shRNA expression vector HU Songquan, YANG Hui, WANG Peng (Department of Anesthesiology,Tongji Hospital,Tongji Medical Collehe,Huazhong University of Science Technology, WuHan 430030) Abstract: 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. Sebusquantly the DNA segments were cloned into pGenesil-1.2 vectors respectively. At last, the plasmids were identified by restriction analysis and sequencing test. Results： 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. Conclusion The DOR-shRNA expression vectors were constructed successfully and could be used in RNAi’s study on morphine tolerance. Keywords: delta opoiod receptor; RNA interference; morphine tolerance; vector constrcution 0 引言 阿片类药物长期应用于慢性疼痛病人将导致药物耐受，影响治疗效果并增加副作用，其 发生机制可能与 Delta 阿片受体（δ-opioid receptor, DOR）在细胞膜表达上调有关，并且受 ，2] 高效且特异性地抑制靶基因的表达。本研拟究构建特异性抑制大鼠 Delta 阿片受体的短发夹 RNA 真核表达载体，为以后通过 RNAi 技术进一步研究 Delta 阿片受体