• 昆明市第一人民醫(yī)院暨昆明醫(yī)學院附屬甘美醫(yī)院肝膽胰一科，云南省器官移植研究所肝移植研究中心(云南昆明 650011);

目的克隆大鼠galectin-9基因全長cDNA，構(gòu)建含大鼠galectin-9基因的重組腺病毒載體，并予以鑒定。方法從大鼠的肝臟組織中用RT-PCR的方法克隆擴增大鼠galectin-9基因，再定向插入到帶NotⅠ和HindⅢ酶切的pDC316-GFP穿梭質(zhì)粒中，獲得穿梭質(zhì)粒pDC316-GFP-galectin-9。經(jīng)PCR、NotⅠ和HindⅢ酶切及測序鑒定后，用脂質(zhì)體將穿梭質(zhì)粒pDC316-GFP-galectin-9與腺病毒骨架質(zhì)粒pBHGlox△E1.3Cre共轉(zhuǎn)染HEK-293細胞。經(jīng)位點特異性重組獲得含目的基因的重組腺病毒Ad5-galectin-9，行PCR鑒定，經(jīng)HEK-293細胞擴增及純化制備高滴度病毒液，用細胞培養(yǎng)方法測定病毒TCID50滴度。結(jié)果 PCR、酶切及測序證實穿梭質(zhì)粒構(gòu)建正確，PCR鑒定證實大鼠galectin-9基因重組腺病毒載體構(gòu)建正確，病毒的感染滴度為1.4×109 U/ml。結(jié)論成功構(gòu)建了含大鼠galectin-9基因的重組腺病毒表達載體，為進一步研究大鼠galectin-9基因的功能奠定了基礎(chǔ)。

引用本文： 李來邦,李立,冉江華,趙永恒,張升寧,劉靜,李鑄,蔣益舟. 大鼠galectin-9基因重組腺病毒載體的構(gòu)建和鑒定. 中國普外基礎(chǔ)與臨床雜志, 2011, 18(2): 131-136. doi: 復制

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  1. 1. Hsu DK, Liu FT. Regulation of cellular homeostasis by galectins [J]. Glycoconj J, 2004, 19(79): 507515.
  2. 2. Rabinovich GA, Toscano MA, Ilarregui JM, et al. Shedding light on the immunomodulatory properties of galectins: novel regulators of innate and adaptive immune responses [J]. Glycoconj J, 2004, 19(79): 565573.
  3. 3. Wang F, He W, Zhou H, et al. The Tim3 ligand galectin9 negatively regulates CD8+ alloreactive T cell and prolongs survival of skin graft [J]. Cell Immunol, 2007, 250(12): 6874.
  4. 4. Zhu C, Anderson AC, Schubart A, et al. The Tim3 ligand galectin9 negatively regulates T helper type 1 immunity [J]. Nat Immunol, 2005, 6(12): 12451252.
  5. 5. LaBarre DD, Lowy RJ. Improvements in methods for calculating virus titer estimates from TCID50 and plaque assays [J]. J Virol Methods, 2001, 96(2): 107126.
  6. 6. Wada J, Kanwar YS. Identification and characterization of galectin9, a novel βgalactosidebinding mammalian lectin [J]. J Biol Chem, 1997, 272(9): 60786086.
  7. 7. Türeci O, Schmitt H, Fadle N, et al. Molecular definition of a novel human galectin which is immunogenic in patients with Hodgkin’s disease [J]. J Biol Chem, 1997, 272(10): 64166422.
  8. 8. 楊志明, 伍曉汀, 何濤. Galectin3與腫瘤轉(zhuǎn)移 [J]. 中國普外基礎(chǔ)與臨床雜志, 2005, 12(3): 310313.
  9. 9. 陳耿臻, 韓慧, 許銘炎, 等. ADVtk重組腺病毒的構(gòu)建及檢測 [J]. 中國普外基礎(chǔ)與臨床雜志, 2010, 17(7): 703706.
  10. 10. 曹永寬, 莫永炎, 劉志峰, 等. 人AWP1重組腺病毒的構(gòu)建及其在人血管內(nèi)皮細胞中的表達和定位 [J]. 中國普外基礎(chǔ)與臨床雜志, 2009, 16(12): 987990.
  11. 11. 韓麗英, 葉明珠, 李荷蓮, 等. 多藥耐藥基因轉(zhuǎn)染對人胎盤源性間充質(zhì)干細胞生物學特性影響研究 [J]. 中華醫(yī)學雜志, 2009, 89(39): 27932796.
  12. 12. 張明滿, 李德華, 茍興華, 等. 靶向肝癌細胞的重組腺相關(guān)病毒載體的構(gòu)建 [J]. 中國普外基礎(chǔ)與臨床雜志, 2005, 12(2): 142146.
  13. 13. 陳剛, 韓江, 劉永康, 等. 攜帶IL13基因大鼠肝干細胞系的建立 [J]. 中國普外基礎(chǔ)與臨床雜志, 2009, 16(4): 276280.
  14. 14. Bessis N, GarciaCozar FJ, Boissier MC. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms [J]. Gene Ther, 2004, 11 Suppl 1: S10S17.
  15. 15. Snyder RO, Miao C, Meuse L, et al. Correction of hemophilia B in canine and murine models using recombinant adenoassociated viral vectors [J]. Nat Med, 1999, 5(1): 6470.
  16. 16. Barquinero J, Eixarch H, PérezMelgosa M. Retroviral vectors: new applications for an old tool [J]. Gene Ther, 2004, 11 Suppl 1: S3S9.
  17. 17. Pandori M, Hobson D, Sano T. Adenovirusmicrobead conjugates possess enhanced infectivity: a new strategy for localized gene delivery [J]. Virology, 2002, 299(2): 204212.
  18. 18. Nász I, Adám E. Recombinant adenovirus vectors for gene therapy and clinical trials [J]. Acta Microbiol Immunol Hung, 2001, 48(34): 323348.
  19. 19. Adám E, Nász I. Adenovirus vectors and their clinical application in gene therapy [J]. Orv Hetil, 2001, 142(38): 20612070.
  20. 20. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery [J]. Curr Opin Biotechnol, 1999, 10(5): 440447.
  21. 21. Morsy MA, Gu M, Motzel S, et al. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene [J]. Proc Natl Acad Sci USA, 1998, 95(14): 78667871.
  22. 22. Gilgenkrantz H, Duboc D, Juillard V, et al. Transient expression of genes transferred in vivo into heart using firstgeneration adenoviral vectors: role of the immune response [J]. Hum Gene Ther, 1995, 6(10): 12651274.
  23. 23. Tripathy SK, Black HB, Goldwasser E, et al. Immune responses to transgeneencoded proteins limit the stability of gene expression after injection of replicationdefective adenovirus vectors [J]. Nat Med, 1996, 2(5): 545550.
  24. 24. He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses [J]. Proc Natl Acad Sci USA, 1998, 95(5): 25092514.
  25. 25. Ng P, Parks RJ, Cummings DT, et al. A highefficiency Cre/loxPbased system for construction of adenoviral vectors [J]. Hum Gene Ther, 1999, 10(16): 26672672.