携带NMU2R shRNA重组腺相关病毒载体的构建及其评价
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摘要
构建稳定表达NMU2R shRNA的重组腺相关病毒载体,制备并纯化靶向性沉默NMU2R表达的高滴度重组腺相关病毒。首先设计合成NMU2R shRNA,退火形成双链与p AAV-ZsGreen-shRNA质粒Bam H I和Hind III双酶切产物相连接构建形成质粒p AAV-ZsGreen-r NMU2R shRNA,经双酶切和测序鉴定证实,重组质粒克隆正确,将重组质粒转染到AAV-293包装细胞中包装成腺相关病毒载体,成功制备重组腺相关病毒r AAV5-Zs Green-r NMU2R shRNA,经测定纯化所得r AAV5病毒滴度为1×10~(12)vg/m L。最后将r AAV5-Zs Green-r NMU2R shRNA感染大鼠嗜铬细胞瘤细胞PC12,检测NMU2R shRNA基因沉默效果,利用空病毒载体p AAV-ZsGreen-shRNA作对照,Real-time PCR及Western Blot方法测得NMU2R mRNA及蛋白表达水平与对照组相比显著下降。综上,成功构建了高滴度携带NMU2R shRNA重组腺相关病毒载体r AAV5-Zs Green-r NMU2R shRNA。
To construct the recombinant adeno-associated virus vector carrying neuromedin U 2 receptor( NMU2 R) short hairpin RNA( shRNA) for preparation of high-titer viruses,hairpin RNA was designed to target NMU2R rat mRNA,and was synthesized and cloned into p AAV-ZsGreen-shRNA plasmid which was double digested by Bam H I and Hind III. The presence of the target sequence in the plasmid p AAV-ZsGreen-r NMU2R shRNA was confirmed by enzyme digestion,PCR and DNA sequencing analysis. And then the plasmid was transfected into AAV 293 cells. AAV 293 cells expression NMU2R shRNA were obtained and subsequently infected AAV packaging system to package the r AAV5-Zs Green-r NMU2R shRNA. After purification,the functional and infectious virus was obtained and the titer of virus was 1 × 10~(12) vg/m L. Real-time PCR and western blot methods were used to detect the expression of NMU2R after infection with PC12 cells,and the empty viral vector p AAV-ZsGreen-shRNA was used as control. The results indicated that the expression of NMU2R mRNA and protein was decreased significantly after infection for 24 h and 48 h as compared with that of the control. In summary,a high-titer recombinant adeno-associated virus-5 vector carrying NMU2 R shRNA has been constructed successfully.
To construct the recombinant adeno-associated virus vector carrying neuromedin U 2 receptor( NMU2 R) short hairpin RNA( shRNA) for preparation of high-titer viruses,hairpin RNA was designed to target NMU2R rat mRNA,and was synthesized and cloned into p AAV-ZsGreen-shRNA plasmid which was double digested by Bam H I and Hind III. The presence of the target sequence in the plasmid p AAV-ZsGreen-r NMU2R shRNA was confirmed by enzyme digestion,PCR and DNA sequencing analysis. And then the plasmid was transfected into AAV 293 cells. AAV 293 cells expression NMU2R shRNA were obtained and subsequently infected AAV packaging system to package the r AAV5-Zs Green-r NMU2R shRNA. After purification,the functional and infectious virus was obtained and the titer of virus was 1 × 10~(12) vg/m L. Real-time PCR and western blot methods were used to detect the expression of NMU2R after infection with PC12 cells,and the empty viral vector p AAV-ZsGreen-shRNA was used as control. The results indicated that the expression of NMU2R mRNA and protein was decreased significantly after infection for 24 h and 48 h as compared with that of the control. In summary,a high-titer recombinant adeno-associated virus-5 vector carrying NMU2 R shRNA has been constructed successfully.
引文
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[14]NISHIZAWA N,KANEMATSU-YAMAKI Y,FUNATA M,et al.A potent neuromedin U receptor 2-selective alkylated peptide[J].Bioorganic&Medicinal Chemistry Letters,2017,27(20):4626-4629.
[2]DALBOGE L S,PEDERSEN S L,SECHER T,et al.Neuromedin Uinhibits food intake partly by inhibiting gastric emptying[J].Peptides,2015,69:56-65.
[3]郑旭煦,欧阳克清,高虹,等.神经介素NMU2R受体激动剂的高通量筛选模型研究[J].中国药学杂志,2004,29(3):185-188.
[4]ZHENG X X,GUO L X,WANG D Q,et al.p-Synephrine:a novel agonist for neuromedin U2 receptor[J].Biological&Pharmacentical Bulletin,2014,37(5):764-770.
[5]FUNG-BERMAN A,MYERS A.Citrus aurantium,an ingredient of dietary supplements marketed for weight loss:current status of clinical and basic research[J].Experimental Biology and Medicine,2004,229:698-704.
[6]HOWARD A D,WANG R,PONG S S,et al.Identification of receptors for neuromedin U and its role in feeding[J].Nature,2000,406(6791):70-74.
[7]MARUYAMA K,KAIYA H,MIYAZATO M,et al.Isolation and characterization of two c DNAs encoding the neuromedin U receptor from goldfish brain[J].Journal of Neuroendocrinology,2011,23(3):282-291.
[8]YANG G,SU J,YAO Y,et al.Distribution of neuromedin S and its receptor NMU2R in pigs[J].Researoh in Veterinary Science,2012,92(2):180-186.
[9]KAISHO T,NAGAI H,ASAKAWA T,et al.Effects of peripheral administration of a Neuromedin U receptor 2-selective agonist on food intake and body weight in obese mice[J].International Journal of Obesity(Lond),2017,41(12):1790-1797.
[10]IVANOV T R,LAWRENCE C B,STANLEY P J,et al.Evaluation of neuromedin U actions in energy homeostasis and pituitary function[J].Endocrinology,2002,143(10):3813-3821.
[11]WREN A M,SMALL C J,ABBOTT C R,et al.Hypothalamic actions of neuromedin U[J].Endocrinology,2002,143(11):4227-4234.
[12]EGECIOGLU E,PLOJ K,XU X,et al.Central NMU signaling in body weight and energy balance regulation:evidence from NMUR2deletion and chronic central NMU treatment in mice[J].American Journal of Physiology Endocrinology and Metabolism,2009,297(3):E708-716.
[13]TAKIZAWA T,GEMMA A,UI-TEI K,et al.Basic and clinical studies on functional RNA molecules for advanced medical technologies[J].Journal of Nippon Medical School,2010,77(2):71-79.
[14]NISHIZAWA N,KANEMATSU-YAMAKI Y,FUNATA M,et al.A potent neuromedin U receptor 2-selective alkylated peptide[J].Bioorganic&Medicinal Chemistry Letters,2017,27(20):4626-4629.