Construction and Identification of Recombinant Adenovirus Vector of Rat Galectin-9 Gene_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LI Laibang , LI Li , RAN Jianghua , ZHAO Yongheng , ZHANG Shengning , LIU Jing , LI Zhu , JIANG Yizhou.

The First Department of Hepatopancreatobiliary Surgery, The Affiliated Ganmei Hospital of Kunming Medical College and The First People’s Hospital of Kunming City and Liver Transplantation Center of Organ Transplantation Institute of Yunnan Province, Kunming 650011, Yunnan Province, China;

Corresponding author：

Keywords：

Rat; Galectin-9 gene; Gene clone; Adenovirus vector; RT-PCR

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ObjectiveTo clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene. MethodsThe galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ. The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) was generated by sitespecific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in HEK-293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. ResultsConstruction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 U/ml. ConclusionRecombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) is successfully constructed, which provides the foundation of further research on the function of galectin-9 gene.

Citation： LI Laibang,LI Li,RAN Jianghua,ZHAO Yongheng,ZHANG Shengning,LIU Jing,LI Zhu,JIANG Yizhou.. Construction and Identification of Recombinant Adenovirus Vector of Rat Galectin-9 Gene. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2011, 18(2): 131-136. doi: Copy

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1. Hsu DK, Liu FT. Regulation of cellular homeostasis by galectins [J]. Glycoconj J, 2004, 19(79): 507515.
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. 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. 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. 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. 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. 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. 杨志明, 伍晓汀, 何涛. Galectin3与肿瘤转移 [J]. 中国普外基础与临床杂志, 2005, 12(3): 310313.
9. 陈耿臻, 韩慧, 许铭炎, 等. ADVtk重组腺病毒的构建及检测 [J]. 中国普外基础与临床杂志, 2010, 17(7): 703706.
10. 曹永宽, 莫永炎, 刘志峰, 等. 人AWP1重组腺病毒的构建及其在人血管内皮细胞中的表达和定位 [J]. 中国普外基础与临床杂志, 2009, 16(12): 987990.
11. 韩丽英, 叶明珠, 李荷莲, 等. 多药耐药基因转染对人胎盘源性间充质干细胞生物学特性影响研究 [J]. 中华医学杂志, 2009, 89(39): 27932796.
12. 张明满, 李德华, 苟兴华, 等. 靶向肝癌细胞的重组腺相关病毒载体的构建 [J]. 中国普外基础与临床杂志, 2005, 12(2): 142146.
13. 陈刚, 韩江, 刘永康, 等. 携带IL13基因大鼠肝干细胞系的建立 [J]. 中国普外基础与临床杂志, 2009, 16(4): 276280.
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. 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. 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. 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. 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. Adám E, Nász I. Adenovirus vectors and their clinical application in gene therapy [J]. Orv Hetil, 2001, 142(38): 20612070.
20. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery [J]. Curr Opin Biotechnol, 1999, 10(5): 440447.
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. 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. 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. 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. 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Identification of Recombinant Adenovirus Vector of Rat Galectin-9 Gene

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