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重组人RAMP-1基因腺病毒载体的构建及鉴定 被引量：5

Construction and identification of human RAMP1 gene recombinant adenovirus
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摘要 目的构建携带人受体活性修饰蛋白-1(RAMP1)基因的腺病毒表达载体,为进一步深入研究RAMP1的功能奠定基础。方法通过基因工程技术将RAMP1基因克隆到穿梭质粒pShuttle-GFP-CMV中;I-Ceu I+I-Sce I双酶切穿梭质粒pShuttle-GFP-CMV-RAMP1,将回收的GFP-CMV-RAMP1片段,亚克隆至腺病毒骨架载体pAdxsi得到重组腺病毒质粒;重组腺病毒质粒酶切线性化后应用脂质体法转染293细胞进行包装扩增,得到重组腺病毒Ad-RAMP1并进行PCR鉴定及滴度测定。结果构建的重组穿梭质粒pShuttle-GFP-CMV-RAMP1双酶切后,得到0.8kb(RAMP1)和5.1kb(pShuttle-GFP-CMV)两个片段;重组腺病毒质粒pAdxsi-GFP-CMV-RAMP1用XhoI酶切得到7个片段,而作为对照的空腺病毒质粒只得到6个片段;重组腺病毒Ad-RAMP1 PCR鉴定可见阳性扩增条带;病毒滴度检测达4.5×1011PFU/ml。结论成功构建了携带人RAMP1基因的重组腺病毒载体,为进一步研究RAMP1的功能研究奠定了基础。 Objective To construct and identify human RAMP1 recombinant adenovirus. Methods Human RAMP1 gene segment was subcloned into pShuttle-GFP-CMV. PShuttle-CMV-RAMP1 was digested by I-Ceu I ＋I-Sce I double enzyme and subcloned into adenovirus backbone vector to form recombinant adenovirus plasmid. Recombinant adenovirus plasmid was transfected into 293 cell lines by liposome. Human RAMP1 recombinant adenovirus was detected by polymerase chain reaction, the titer of virus was analyzed. Results Cloned sequence 0.8kb （RAMP1） and 5.1kb （pShuttle-GFP-CMV）was obtained by double enzyme after RAMP1 cDNA segment was cloned into pShuttle-GFP-CMV. DNA sequencing results indicated that the clone location was correct. Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after digested by XhoI RAMPlcDNA （0.8kb） was amplified by PCR with virus titer of 4.5×10^11PFU/ml. Conclusions The recombinant adenovirus containing human RAMP1 gene was established successfully. The wiU be helpful for further study of the function of human RAMP1.
作者 石蓓 宋付凯 赵然尊 刘志江 龙仙萍 盛瑾
机构地区 遵义医学院附属医院心内科
出处 《遵义医学院学报》 2010年第4期330-332,共3页 Journal of Zunyi Medical University
基金 国家自然科学基金项目(30860100)
关键词 EGFP RAMP1 腺病毒表达载体 基因重组 receptor activity modifying protein-1 recombinant adenovirus construction
分类号 R512.36 [医药卫生—内科学]
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参考文献8

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同被引文献45

  • 1中华人民共和国科学技术部.关于善待实验动物的指导性意见.2006.09-30
  • 2Marx SO,Totary-Jain H,Marks AR.Vascular smooth muscle cell proliferation in restenosis.Circ Cardiovasc Interv.2011;4(1):104-111.
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  • 4Zhang Z,Winborn CS,de Prado B M,et al.Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein-1 in the trigeminal ganglion.J Neurosci.2007;27(10):2693-2703.
  • 5Poyner DR,Sexton PM,Marshall I,et al.International Union of Pharmacology.XXXII.The mammalian calcitonin gene-related peptides,adrenomedullin,amylin,and calcitonin receptors.Pharmacol Rev.2002;54(2):233-246.
  • 6Qin XP,Ye F,Hu CP,et al.Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells.Eur J Pharmacol.2004;488(1-3):45-49.
  • 7Chattergoon NN,D'Souza FM,Deng W,et al.Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells.Am J Physiol Lung Cell Mol Physiol.2005;288(1):L202-L211.
  • 8Zhang Z,Dickerson IM,Russo AF.Calcitonin gene-related peptide receptor activation by receptor activity-modifying protein-1 gene transfer to vascular smooth muscle cells.Endocrinology.2006;147(4):1932-1940.
  • 9Tsujikawa K,Yayama K,Hayashi T,et al.Hypertension and dysregulated proinflammatory cytokine production in receptor activity-modifying protein 1-deficient mice.Proc Natl Acad Sci U S A.2007;104(42):16702-16707.
  • 10Chrissobolis S,Zhang ZM,Lynch CM,et al.Receptor activity modifying protein-1 (RAMP1) overexpression selectively enhances calcitonin gene-related peptide-induced vasodilation.The FASEB Journal.2008;22:1151-1153.

引证文献5

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遵义医学院学报
2010年 第4期

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