抑制coronin-1基因表达的siRNA质粒载体的构建及筛选研究
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摘要
目的:构建特异性抑制Coronin-1基因表达的siRNA表达载体,并筛选出具有最高抑制效率的质粒载体。
方法:提取鼠巨噬细胞总RNA,RT-PCR扩增Coronin-1基因目标序列,将其连接到pSEB-HUS真核表达质粒上,重组质粒经酶切和DNA测序鉴定后命名为pSEB-HUS-C。将设计、合成的3条siRNA及阴性对照分别克隆入pSEB-HUS-C ,构建重组pSEB-HUS-C1、pSEB-HUS-C2、pSEB-HUS-C3及pSEB-HUS-CN质粒。将干涉质粒瞬时转染A549细胞,通过绿色荧光信号观察不同重组质粒对Coronin-1表达的影响。最后用实时定量PCR和Western blot法检测其对Coronin-1基因表达的抑制效率。
结果:经双酶切及测序证实,所构建siRNA表达载体目的基因大小、序列与预期相符。经瞬时转染A549细胞后,其中的pSEB-HUS-C3能明显抑制Coronin-1 mRNA的表达和Coronin-1蛋白的合成,抑制率分别是75.9%和75.1%。
结论:成功构建并筛选出高效、特异性抑制Coronin-1表达的siRNA表达载体,为进一步研究Coronin-1在巨噬细胞中的作用奠定了基础。
Objective: To construct the plasmid vectors of siRNA which can specifically suppress the expression of coronin-1 gene and select the most effective one for further study.
Methods: The cDNA of coronin-1 was amplified from the total RNA of macrophage by RT-PCR, then it was inserted into pSEB-HUS vector. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids were named pSEB-HUS-C. 3 synthesized siRNAs were cloned into pSEB-HUS-C respectively, in order to construct the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids, which were designed for Coronin-1 as target gene. After these plasmids was transiently transfected into A549, the inhibition level of Coronin-1 in A549 cells was detected by RT-PCR, Real time PCR and Western blot.
Result: The recombinant plasmids were confirmed by double-enzyme digestion and DNA sequencing. The expression of Coronin-1 mRNA and Coronin-1 protein of pSEB-HUS-C3 group decreased significantly and the inhibition rate was 75.9% and 75.1% respectively.
Conclusion: Specific siRNA interference plasmid vector targeted to coronin-1 gene mRNA is successfully constructed, which provides a good basis for further research on the function of Coronin-1 against tuberculosis
方法:提取鼠巨噬细胞总RNA,RT-PCR扩增Coronin-1基因目标序列,将其连接到pSEB-HUS真核表达质粒上,重组质粒经酶切和DNA测序鉴定后命名为pSEB-HUS-C。将设计、合成的3条siRNA及阴性对照分别克隆入pSEB-HUS-C ,构建重组pSEB-HUS-C1、pSEB-HUS-C2、pSEB-HUS-C3及pSEB-HUS-CN质粒。将干涉质粒瞬时转染A549细胞,通过绿色荧光信号观察不同重组质粒对Coronin-1表达的影响。最后用实时定量PCR和Western blot法检测其对Coronin-1基因表达的抑制效率。
结果:经双酶切及测序证实,所构建siRNA表达载体目的基因大小、序列与预期相符。经瞬时转染A549细胞后,其中的pSEB-HUS-C3能明显抑制Coronin-1 mRNA的表达和Coronin-1蛋白的合成,抑制率分别是75.9%和75.1%。
结论:成功构建并筛选出高效、特异性抑制Coronin-1表达的siRNA表达载体,为进一步研究Coronin-1在巨噬细胞中的作用奠定了基础。
Objective: To construct the plasmid vectors of siRNA which can specifically suppress the expression of coronin-1 gene and select the most effective one for further study.
Methods: The cDNA of coronin-1 was amplified from the total RNA of macrophage by RT-PCR, then it was inserted into pSEB-HUS vector. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids were named pSEB-HUS-C. 3 synthesized siRNAs were cloned into pSEB-HUS-C respectively, in order to construct the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids, which were designed for Coronin-1 as target gene. After these plasmids was transiently transfected into A549, the inhibition level of Coronin-1 in A549 cells was detected by RT-PCR, Real time PCR and Western blot.
Result: The recombinant plasmids were confirmed by double-enzyme digestion and DNA sequencing. The expression of Coronin-1 mRNA and Coronin-1 protein of pSEB-HUS-C3 group decreased significantly and the inhibition rate was 75.9% and 75.1% respectively.
Conclusion: Specific siRNA interference plasmid vector targeted to coronin-1 gene mRNA is successfully constructed, which provides a good basis for further research on the function of Coronin-1 against tuberculosis
引文
[1] Floyd K, Blanc L, Ravigione M, et al. Resources required for global tuberculosis control[J]. Science, 2002,295(5562):2040-2041.
[2] Vergne I, Chua J, Singh SB, et al. Cell biology of mycobacterium tuberculosis phagosome[J]. Annu Rev Cell Dev Biol, 2004,20:367-394.
[3] Veronique Kiermer. Focus on RNA interference[J]. Nature Methods, 2006,3:669-669.
[4] Mello CC, Conte D Jr. Revealing the world of RNA interference[J]. Nature, 2004,431(7006):338-342.
[5] Putral LN, Gu W, McMillan NA. RNA interference for the treatment of cancer[J]. Drug News Perspect, 2006,19(6):317-324.
[6]张泳,谷仲平,周勇安,等. RNAi沉默EVGF的表达及其治疗肺癌的初步研究[J].分子与细胞免疫学杂志,2009,25(4):341-343.
[7] Anne Lise K, Hestvik, Zakaria Hmama, et al. Mycobacterial manipulation of the host cell. FEMS Microbiol Rev. 2005,9:1041-1050.
[8]李俊明,朱道银.结核分枝杆菌的感染与巨噬细胞的凋亡[J].国际检验医学杂志, 2006,27(1):65-68.
[9] Ferrari G, Langen H, Naito M, et al. A coat protein on phagosomes involved in the intracellular survival of mycobacteria[J]. Cell, 1999,97(4): 435-447.
[10] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998,391(6669):806-811.
[11] J.萨姆布鲁克, D.W.拉塞尔.分子克险实验指南[M].第3版,北京:科学出版社, 2002,1564-1594.
[12]吴雄文,梁智辉.实用免疫学实验技术[M].武汉:湖北科学技术出版社, 2002,43-73.
[13]孙树汉.基因工程原理与方法[M].北京:人民军医出版社, 2002,116-123.
[14]张维铭.现代分子生物学实验手册[M].北京:科学出版社, 2003,244-310.
[15] J.萨姆布鲁克, D.W.拉塞尔.分子克险实验指南[M].第3版,北京:科学出版社, 2002,87-98.
[16]张维铭.现代分子生物学实验手册[M].北京:科学出版社, 2003,213-218.
[17] Neer E.J, Schmicit C.J, Nambudripad R, Smith T.F. The ancient regulatory-protein family of WD-repeat proteins[J]. Nature, 371:297-300.
[18] King S.M, Patel-king R.S, Wilkerson C.G, Witman G.B. The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein[J]. Cell Biol, 131:399-409.
[19] Shriayama M, Zachariae W, Ciosk R, Nasmyth K. The Polo-like kinase Cdc5p and WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae[J]. EMBO J, 17:1336-1349.
[20] Pryer N.K, Salama N.R, Schekman R, Kaiser C.A. Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro[J]. Cell Biol, 120:865-875.
[21] Li Q, Suprenant K.A. Molecular characterization of the 77-kDa echinoderm microtubule-associated protein[J]. Biol. Chem, 269:31777-31784.
[22] Paul C.P, Good P.D, Winter I, Engelke D.R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol, 2000,20:505-508.
[23] Luo Q, Song W.X, et al. Selection and validati optimal siRNA target sites for RNAi-mediated gene silencing[J]. Gene, 2007,395(1-2):160-169.
[24] Montgomery R.A, Dallman M.J. Semi-quantitative polymerase chain reaction analysis of cytokine and cytokine receptor gene expreasion during thymic ontogeny[J]. Cytokine, 1997,97:717-726.
[25] Wall S.J, Edward D.R, Quantitative reverse transcription-polymerase chain reaction(RT-PCR): A comparison of prime-dropping, competitive, and real-time RT-PCRa[J]. Anal Biochem, 2002,300(2):269-273.
[26] Schmittgen T.D, Zakrajsek B.A, Mills A.G, et al. Quantitatitive reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and realtime methods[J]. Anal Biochem, 2000,285(2):194-204.
[27] Whitcombe D, Theaker J, Guy S P, et al. Detection of PCR products using self-probing amplicons and fluorescence[J]. Nat Biotechnol, 1999,17(8):804-807.
[28] Mackay I M, Arden K E, Nitsche A, Real-time PCR in virology[J]. Nucleic Acids Res, 2002,30(60):1292-1305.
[29] Heid C.A, Stevens J, Livak J.K, et al. Real time quantitative PCR[J]. Genome Res, 1996,6:986-994.
[30] Luo Q, Kang Q, Song WX, et al. Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing[J]. Gene, 2007,395(1-2):160-169.
1. Floyd K, Blanc L, Ravigione M, et al. Resources required for global tuberculosis control[J]. Science, 2002, 295(5562): 2040-2041.
2. Vergne I, Chua J, Singh SB, et al. Cell biology of mycobacterium tuberculosis phagosome[J]. Annu Rev Cell Dev Biol, 2004, 20: 367-394.
3. Christopher D, Williams B.G, et al. Erasing the World’s Slow Stain:Strategies to Best Multidrug-Resistant Tuberculosis[J]. Science, 2002, 295(5562): 2042-2046.
4. Alastair J, Woo D. Drug therapy[J]. N EngL J MeD, 1993, 329:784-791.
5.周光炎.免疫学原理.上海:上海科学技术文献出版社, 2007, 3: 87 -89.
6. Janeway CA, Travers P, Walport M et al. Immunobiology[M]. Garland Scie Publ, 2005, 747-750.
7. Roy E, Brennan J, Jolles S, Lowrie DB. Beneficial effect of anti-interleukin-4 antibody when administered in a marine model of tuberculosis infection[J]. Tuberculosis. 2008, 88: 197-202.
8. Hohesise LG, Chan BKM, Chan CHS, et al. En dobronchial Tuberculosis:Diagnostic features and therapeutic outcome[J]. Res Prie MeD, 1994, 88: 593-597.
9. Sanehez FO, Jaime I, Rodriguez, et al.Immune res Ponsiveness and Lympokine Production in patients with tube rculosis and healthy controls[J]. Infeetimmun, 1994, 62: 5673-5678.
10.Veronique Kiermer. Focus on RNA interference[J]. Nature Methods, 2006, 3: 669-669.
11. Sundaram urthy V, Pieters J. Interactions of pathogenic mycobacteria with host macrophages[J]. Microbes and Infection, 2007, 9: 1671-1679.
12. Kuijl C, Savage NDL, Marsman M, et al. Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1. Nature, 2007,450:725-732.
13. Rupper AC, Rodriguez-Paris JM, Grove BD, et al. PI 10-related PI3-Kinases regulate phagosome fusion and phagosomal pH through a PKB, Akt dependent pathway in Dictyostelium[J]. Cell Sci, 2001,114:1283-1295.
[2] Vergne I, Chua J, Singh SB, et al. Cell biology of mycobacterium tuberculosis phagosome[J]. Annu Rev Cell Dev Biol, 2004,20:367-394.
[3] Veronique Kiermer. Focus on RNA interference[J]. Nature Methods, 2006,3:669-669.
[4] Mello CC, Conte D Jr. Revealing the world of RNA interference[J]. Nature, 2004,431(7006):338-342.
[5] Putral LN, Gu W, McMillan NA. RNA interference for the treatment of cancer[J]. Drug News Perspect, 2006,19(6):317-324.
[6]张泳,谷仲平,周勇安,等. RNAi沉默EVGF的表达及其治疗肺癌的初步研究[J].分子与细胞免疫学杂志,2009,25(4):341-343.
[7] Anne Lise K, Hestvik, Zakaria Hmama, et al. Mycobacterial manipulation of the host cell. FEMS Microbiol Rev. 2005,9:1041-1050.
[8]李俊明,朱道银.结核分枝杆菌的感染与巨噬细胞的凋亡[J].国际检验医学杂志, 2006,27(1):65-68.
[9] Ferrari G, Langen H, Naito M, et al. A coat protein on phagosomes involved in the intracellular survival of mycobacteria[J]. Cell, 1999,97(4): 435-447.
[10] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998,391(6669):806-811.
[11] J.萨姆布鲁克, D.W.拉塞尔.分子克险实验指南[M].第3版,北京:科学出版社, 2002,1564-1594.
[12]吴雄文,梁智辉.实用免疫学实验技术[M].武汉:湖北科学技术出版社, 2002,43-73.
[13]孙树汉.基因工程原理与方法[M].北京:人民军医出版社, 2002,116-123.
[14]张维铭.现代分子生物学实验手册[M].北京:科学出版社, 2003,244-310.
[15] J.萨姆布鲁克, D.W.拉塞尔.分子克险实验指南[M].第3版,北京:科学出版社, 2002,87-98.
[16]张维铭.现代分子生物学实验手册[M].北京:科学出版社, 2003,213-218.
[17] Neer E.J, Schmicit C.J, Nambudripad R, Smith T.F. The ancient regulatory-protein family of WD-repeat proteins[J]. Nature, 371:297-300.
[18] King S.M, Patel-king R.S, Wilkerson C.G, Witman G.B. The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein[J]. Cell Biol, 131:399-409.
[19] Shriayama M, Zachariae W, Ciosk R, Nasmyth K. The Polo-like kinase Cdc5p and WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae[J]. EMBO J, 17:1336-1349.
[20] Pryer N.K, Salama N.R, Schekman R, Kaiser C.A. Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro[J]. Cell Biol, 120:865-875.
[21] Li Q, Suprenant K.A. Molecular characterization of the 77-kDa echinoderm microtubule-associated protein[J]. Biol. Chem, 269:31777-31784.
[22] Paul C.P, Good P.D, Winter I, Engelke D.R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol, 2000,20:505-508.
[23] Luo Q, Song W.X, et al. Selection and validati optimal siRNA target sites for RNAi-mediated gene silencing[J]. Gene, 2007,395(1-2):160-169.
[24] Montgomery R.A, Dallman M.J. Semi-quantitative polymerase chain reaction analysis of cytokine and cytokine receptor gene expreasion during thymic ontogeny[J]. Cytokine, 1997,97:717-726.
[25] Wall S.J, Edward D.R, Quantitative reverse transcription-polymerase chain reaction(RT-PCR): A comparison of prime-dropping, competitive, and real-time RT-PCRa[J]. Anal Biochem, 2002,300(2):269-273.
[26] Schmittgen T.D, Zakrajsek B.A, Mills A.G, et al. Quantitatitive reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and realtime methods[J]. Anal Biochem, 2000,285(2):194-204.
[27] Whitcombe D, Theaker J, Guy S P, et al. Detection of PCR products using self-probing amplicons and fluorescence[J]. Nat Biotechnol, 1999,17(8):804-807.
[28] Mackay I M, Arden K E, Nitsche A, Real-time PCR in virology[J]. Nucleic Acids Res, 2002,30(60):1292-1305.
[29] Heid C.A, Stevens J, Livak J.K, et al. Real time quantitative PCR[J]. Genome Res, 1996,6:986-994.
[30] Luo Q, Kang Q, Song WX, et al. Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing[J]. Gene, 2007,395(1-2):160-169.
1. Floyd K, Blanc L, Ravigione M, et al. Resources required for global tuberculosis control[J]. Science, 2002, 295(5562): 2040-2041.
2. Vergne I, Chua J, Singh SB, et al. Cell biology of mycobacterium tuberculosis phagosome[J]. Annu Rev Cell Dev Biol, 2004, 20: 367-394.
3. Christopher D, Williams B.G, et al. Erasing the World’s Slow Stain:Strategies to Best Multidrug-Resistant Tuberculosis[J]. Science, 2002, 295(5562): 2042-2046.
4. Alastair J, Woo D. Drug therapy[J]. N EngL J MeD, 1993, 329:784-791.
5.周光炎.免疫学原理.上海:上海科学技术文献出版社, 2007, 3: 87 -89.
6. Janeway CA, Travers P, Walport M et al. Immunobiology[M]. Garland Scie Publ, 2005, 747-750.
7. Roy E, Brennan J, Jolles S, Lowrie DB. Beneficial effect of anti-interleukin-4 antibody when administered in a marine model of tuberculosis infection[J]. Tuberculosis. 2008, 88: 197-202.
8. Hohesise LG, Chan BKM, Chan CHS, et al. En dobronchial Tuberculosis:Diagnostic features and therapeutic outcome[J]. Res Prie MeD, 1994, 88: 593-597.
9. Sanehez FO, Jaime I, Rodriguez, et al.Immune res Ponsiveness and Lympokine Production in patients with tube rculosis and healthy controls[J]. Infeetimmun, 1994, 62: 5673-5678.
10.Veronique Kiermer. Focus on RNA interference[J]. Nature Methods, 2006, 3: 669-669.
11. Sundaram urthy V, Pieters J. Interactions of pathogenic mycobacteria with host macrophages[J]. Microbes and Infection, 2007, 9: 1671-1679.
12. Kuijl C, Savage NDL, Marsman M, et al. Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1. Nature, 2007,450:725-732.
13. Rupper AC, Rodriguez-Paris JM, Grove BD, et al. PI 10-related PI3-Kinases regulate phagosome fusion and phagosomal pH through a PKB, Akt dependent pathway in Dictyostelium[J]. Cell Sci, 2001,114:1283-1295.