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穿梭质粒pCDH1-GFP-HCN 4的构建及HCN 4重组慢病毒的包装 被引量：3

Construction of the shuttle plasmid pCDH1-GFP-HCN 4 and packaging of HCN 4 recombined lentivirus
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摘要 目的将全长约3.6 kb的HCN 4目的基因片段从pCDNA3-HCN 4质粒卸载下来,建立稳定的HCN 4重组慢病毒。方法用Eco RⅠ和XbaⅠ从pCDNA3-HCN 4质粒切下HCN 4目的片段连接到Puc19载体中,再用EcoRⅠ和HindⅢ从质粒Puc19切下目的片段并连接到pcDNA3.1(A)载体中,再用XbaⅠ单酶从质粒pcDNA3.1(A)双切到pCDH1-MCS1-EF1-copGFP中。用XbaⅠ或者Eco RⅠ鉴定,将阳性克隆质粒送上海生工测序,慢病毒的包装参照SBI的Lentivector Expression System操作手册进行。将构建好的慢病毒包装质粒混合物混合后感染293T细胞及心肌样骨髓基质干细胞。结果 (1)XbaⅠ及Eco RⅠ鉴定表明,PCR产物和克隆扩增产物的电泳结果显示该基因的大小约为3.6 kb;阳性克隆质粒测序结果与GENEBANK目的基因HCN 4序列完全相同;构建的目的基因质粒转染293细胞和原代猪骨髓间充质干细胞后,均发现有强烈的绿色荧光出现。(2)构建的慢病毒包装质粒混合物转染293细胞后,发现有强烈的绿色荧光出现,细胞感染率达90%以上。结论 (1)成功将全长HCN 4目的基因片段从pCDNA3-HCN 4质粒卸载下来,成功构建穿梭质粒pCDH1-MCS1-EF1-copGFP。(2)成功建立稳定的HCN 4重组慢病毒,该病毒载体有很高的转染效率。 Objective HCN 4 gene fragment is cloned from plasmid pCDNA3-HCN 4 and inserted into the shuttle plasmid pCDH1-GFP-HCN 4 and constructed HCN 4 recombined lentivirus. Methods HCN 4 gene fragment was cloned from plasmid pCDNA3-HCN 4 and inserted into plasmid Pucl9 by En- zymes Eco R ｜ and Xba I , Then the target gene was cutted and cloned into plasmid pcDNA3.1 （A） by Enzymes Eco R I and Hind m. In the end, HCN 4 gene fragment was cutted into plasmid pCDH1-MCS1 -EFI-copGFP by Enzymes Xba I ~ Double Enzymes Xba I and Eco R I verified approach and analysising the sequence were used to confirm the positive plasmid. HCN 4 recombined lentivirus was constructed ac- cording to operating manual of Lentivector Expression System. pPACKH1- Lentivector Packaging Kit with three plasmids was used to transfect 293 T cells. After 48 hours culturing, GFP （green） was detected bythe fluorescence microscope. Results Confirmed by enzymes Xba I and Eco R I HCN 4 gene fragment was cloned into the plasmid pCDH1-MCS1 -EFI-copGFP and the sequence in the positive plasmid was confirmed by comparison with the published gene bank. The recombined lentivirus was used to transfect 293 T cells and GFP was observed in the transfected 293 ce cieney of infection was above 90 percents. Conclusion cloned from plasmid pCDNA 3-HCN 4 and inserted into The recombined lentivirus was successfully constructed and lls under the fluorescent microscope. The effi- （1） HCN 4 gene fragment was successfully e shuttle plasmid pCDH1-GFP-HCN 4. （2） had high efficiency of infection.
作者 周亚峰 李红霞 蔡思达 程铖 韩莲花 赵欣 杨向军
机构地区 苏州大学附属第一医院心内科 南京大学生命科学学院
出处 《苏州大学学报（医学版）》 CAS 2012年第3期354-358,437,共6页 Suzhou University Journal of Medical Science
基金 国家自然科学基金资助项目(81170174 81070139) 江苏省自然科学基金(BK2011304) 江苏省"科教兴卫"医学重点人才项目(RC2011111)
关键词 HCN4基因 穿梭质粒 慢病毒 HCN 4 gene shuttle plasmid recombined lentivirus
分类号 R318 [医药卫生—生物医学工程] R541-7205 [医药卫生—心血管疾病]
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参考文献15

  • 1Mobley AS, Miller AM, A.raneda RC ,et al. Hyperpolariza- tion-activated cyclic nucleotide-gated channels in olfactory sensory neurons regulate axon extension and glomerular for- mation[J]. J Neurosci, 2010,30(49) :16498 - 16508.
  • 2Herrmann S, Hofmann F, Stieber J, et al. HCN channels in the heart: Lessons from mouse mutants [ J ]. Br J Phar- macol, 2011,5(10) :5376 -5381.
  • 3Baruscotti M, Barbuti A, Bucchi A. The cardiac pacema- ker current [ J]. J Mol Cell Cardiol, 2010,48 ( 1 ) :55 - 64.
  • 4Lau YT, Weng CK, Luo J, et al. Effects of hyperpolariza- tion-activated cyclic nucleotide-gated (HCN) channel blockers on the proliferation and cell cycle progression of embryonic stem cdls [J]. Pflugers Arch, 2011,461 ( 1 ) : 191 - 202.
  • 5Naldini L, Blomer U, Gallay P, et al. In vivo gene deliv- ery and stable transductlon of nondividing cells by a lenti- viral vector [J]. Science, 1996, 272(5259) :263 -267.
  • 6Naldini L, Blomer U, Gage F H, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector [J]. Proc Natl Acad Sci USA, 1996, 92(21) :11382 - 11388.
  • 7Zufferey R, Nagy D, Mandel R J, et al. Multiply attenua- ted lentiviral vector achieved efficient gene delivery in vivo [J]. Nat Biotechnol, 1997, 15(9) : 871 -875.
  • 8Miyoshi H, Takshashi M, Gage F H, et al. Stable and ef- ficient gene transfer into the retina using an HIV-based lentiviral vector [ J]. Proc Natl Acad Sci USA, 1997,94 (19) : 10319 - 10323.
  • 9Castellani S, Conese M. Lentiviral vectors and cystic fibro- sis gene therapy [J]. Viruses, 2010,2(2) :395 -412.
  • 10Lois C, Elizabeth J Hong, Shirley Pease, et al. Germline transmission and tissue-specific expression of transgenes delivered by Lentiviral vectors [ J ]. Science, 2002, 295(5556):868 -872.

同被引文献43

  • 1Quinlan M P, Settleman J. Isoform-specific ras functions in develop- ment and cancer. Future Oncol, 2009; 5(1) :105-116.
  • 2Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer, 2003 ; 3 ( 6 ) :459-465.
  • 3Bar-Sagi D, Hall A. Ras and Rho GTPases: a family reunion. Cell, 2000 ; 103 ( 2 ) :227-238.
  • 4Der C J, Van Dyke T. Stopping ras in its tracks. Cell, 2007; 129 (5) :855-857.
  • 5Tyner J W, Erickson H, Deininger M W, et al. High-throughput se- quencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients. Blood, 2009 ; 113 ( 8 ) : 1749-1755.
  • 6Zuber J, Radtke I, Pardee T S, et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev, 2009; 23 (7) : 877-889.
  • 7Karnoub A E, Weinberg R A. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol, 2008; 9(7) :517-531.
  • 8Lau Y T, Wong C K, Luo J, et al. Effects of hyperpolarization-acti- rated cyclic nucleotide-gated (HCN) channel blockers on the prolif- eration and cell cycle progression of embryonic stem cells. Pflugers Arch, 2011; 461(1):191-202.
  • 9Naldini L, Blomer U, Gage F H, et al. Efficient transfer, integra- tion, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Nail Acad Sci U S A, 1996; 93(21) :11382-11388.
  • 10Zufferey R, Nagy D, Mandel R J, et al. Multiply attenuated lentivi- ral vector achieves efficient gene delivery in vivo. Nat Biotechnol, 1997; 15(9) :871-875.

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苏州大学学报（医学版）
2012年 第3期

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