Objective To construct AWP1 (associated with protein kinase C related kinase 1) recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304. Methods Cloned AWP1 cDNA was inserted into the multiply clone sites (MCS) of plasmid pcDNA3 for adding flag tag, and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV. After identified with restrictional enzymes, plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease PmeⅠ, and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination. After linearized by PacⅠ, the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high-level adenoviruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope (LSCM). Results PCR assay showed that recombinant adenovirus Ad-flag-AWP1 was obtained successfully; and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWP1 recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.
Citation：
CAO Yongkuan,MO Yongyan,LIU Zhifeng,XUE Gang,WANG Peihong,JIANG Yong,TIAN Fuzhou. Construction of Human Flag-AWP1 Recombinant Adenovirus Vector and Its Expression and Localization in Human Vascular Endothelial Cell. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2009, 16(12): 987-990. doi:
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莫永炎, 曹永宽, 刘亚伟, 等. 人His-AWP1融合蛋白表达载体的构建及其在原核生物的表达 [Ｊ]. 中华神经医学杂志， 2003; 2(2): 123-125.
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曹永宽, 莫永炎, 田伏洲, 等. 人AWP1-RFP融合基因重组腺病毒的快速构建及其鉴定 [Ｊ]. 中华神经医学杂志， 2005; 4(5): 434-438.
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汪胜毅, 王玉琦, 符伟国, 等. 白细胞介素-10腺病毒载体制备及其在血管平滑肌细胞的表达 [Ｊ]. 中国普外基础与临床杂志, 2007; 14(6): 660-664.
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陈方, 薛新波, 王从俊, 等. 腺病毒介导mda-7基因联合阿霉素治疗裸鼠移植肝癌的作用 [Ｊ]. 中国普外基础与临床杂志, 2008； 16(9): 673-677.
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曹永宽, 莫永炎, 田伏洲, 等. 人GFP-AWP1融合基因载体的构建及其在293细胞中的表达 [Ｊ]. 第一军医大学学报, 2005; 25（2）: 174-176.
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- 1. Duan W, Sun B, Li TW, et al. Cloning and characterization of AWP1, a novel protein that associates with serine/threonine kinase PRK1 in vivo [Ｊ]. Gene, 2000; 256(1-2): 113-121.
- 2. Amano M, Mukai H, Ono Y, et al. Identification of a putative target for Rho as the serine-threonine kinase protein kinase N [Ｊ]. Science, 1996; 271(5249): 648-650.
- 3. Watanabe G, Saito Y, Madaule P, et al. Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho [Ｊ]. Science, 1996; 271(5249): 645-648.
- 4. Klug A, Schwabe JW. Protein motifs 5. Zinc fingers [Ｊ]. FASEB J, 1995; 9(8): 597-604.
- 5. He KL, Ting AT. A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells [Ｊ]. Mol Cell Biol, 2002; 22(17): 6034-6045.
- 6. 徐明清, 严律南, 苟兴华, 等. 锌指蛋白A20促进同种异体大鼠小体积移植肝再生并抑制急性排斥 [Ｊ]. 中国普外基础与临床杂志, 2008； 15(8): 572-578.
- 7. 莫永炎, 曹永宽, 刘亚伟, 等. 人His-AWP1融合蛋白表达载体的构建及其在原核生物的表达 [Ｊ]. 中华神经医学杂志， 2003; 2(2): 123-125.
- 8. 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.
- 9. 夏冬, 徐亮, 万礼仪, 等. 重组腺病毒介导hTIMP-1对人肝癌细胞系体外增殖的影响 [Ｊ]. 中国普外基础与临床杂志, 2007； 14(4): 409-412.
- 10. 曹永宽, 莫永炎, 田伏洲, 等. 人AWP1-RFP融合基因重组腺病毒的快速构建及其鉴定 [Ｊ]. 中华神经医学杂志， 2005; 4(5): 434-438.
- 11. 汪胜毅, 王玉琦, 符伟国, 等. 白细胞介素-10腺病毒载体制备及其在血管平滑肌细胞的表达 [Ｊ]. 中国普外基础与临床杂志, 2007; 14(6): 660-664.
- 12. 陈方, 薛新波, 王从俊, 等. 腺病毒介导mda-7基因联合阿霉素治疗裸鼠移植肝癌的作用 [Ｊ]. 中国普外基础与临床杂志, 2008； 16(9): 673-677.
- 13. 曹永宽, 莫永炎, 田伏洲, 等. 人GFP-AWP1融合基因载体的构建及其在293细胞中的表达 [Ｊ]. 第一军医大学学报, 2005; 25（2）: 174-176.
