大鼠δ阿片受体基因小发卡RNA及人前脑啡肽原基因双表达慢病毒载体的构建与鉴定

Construction and Identification of Lentivector Encoding Rat δ Opioid Receptor shRNA and Human Preproenkephalin Gene

目的构建大鼠δ阿片受体(delta opioid receptor,DOR)基因小发卡RNA(shRNA)及人前脑啡肽原基因(hu-man preproenkephalin gene,hPPE)双表达慢病毒载体并鉴定。方法根据大鼠DOR mRNA序列设计shRNA并合成包含正、反义靶序列的互补DNA链,退火后插入至shRNA表达载体pENTR/U6vector中,构建shRNA表达质粒,并转化至感受态细胞DH5α,抽提质粒后进行测序。合成hPPE基因序列并插入到表达载体pcDNA3.1(+)中,转化至感受态细胞DH5α,挑取多个单克隆进行测序验证。将hPPE插入已构建成功的pENTR/U6-shRNA载体中,再与慢病毒载体pLenti7.3/V5-DEST重组,构建慢病毒载体pLenti-CMV-hPPE-U6-shRNA并转化DH5α感受态细胞,筛选阳性克隆并测序验证,保留测序验证正确的LR重组质粒。用构建的慢病毒表达载体pLenti-CMV-hPPE-U6-shRNA和包装质粒共转染293T细胞,包装病毒,并测定滴度。结果经测序验证重组质粒pENTR/U6-shRNA、pcDNA3.1(+)-hPPE和慢病毒载体pLenti-CMV-hPPE-U6-shRNA均构建成功。大鼠δ阿片受体基因小发卡RNA及人前脑啡肽原基因双表达慢病毒包装成功,病毒滴度为1×107 TU/mL。结论大鼠DOR基因小发卡RNA及人前脑啡肽原基因双表达慢病毒载体构建成功,为研究DOR和hPPE在吗啡耐受中的作用机制及探求更加合理的镇痛方式奠定了基础。

Objective To construct lentivector encoding rat δ opioid receptor shRNA and human preproenkephalin gene and identify its titer.Methods According to the sequence of rat DOR mRNA,we designed its shRNA sequence and synthesized the sense and antisense oligonucleotides.After annealing,these double DNA strands were cloned to the pENTR/U6 vector and then transformed into competent cell DH5α.The plasmids were extracted and sequenced.hPPE gene sequence was synthesized and inserted into the expression vector pcDNA3.1（＋）,then the recombinant plasmid was transformed into competent cell DH5α,and several positive monoclones were picked for sequencing.hPPE sequence was inserted into the built pENTR/U6 shRNA vector.And then this vector re-combined with lentiviral vector pLenti7.3/V5-DEST to construct lentiviral vector pLenti-CMV-hPPE-U6-shRNA.Lentiviral vector pLenti-CMV-hPPE-U6-shRNA was transformed into DH5α competent cell,positive monoclones were selected and sequenced,and the correct sequencing LR recombinant plasmids were retained.The constructed lentiviral vector pLenti-CMV-hPPE-U6-shRNA and packaging plasmids were co-transfected into 293T cells for viral packaging,virus stock solution was collected and concentrated by ultracentrifugation,and the titer was determined.Results Verified by sequencing,the recombinant plasmid pENTR/U6-shRNA,pcDNA3.1（＋）-hPPE and lentiviral vector pLenti-CMV-hPPE-U6-shRNA were all constructed successfully.Lentivirus encoding rat δ opioid receptor shRNA and human preproenkephalin gene was successfully packaged with the titer of 1×107 TU/mL.Conclusion Lentivirus encoding rat DOR shRNA and human preproenkephalin gene was successfully packaged,which laid the foundation for the study of DOR and hPPE in morphine tolerance mechanism and exploring a more rational way for analgesia.