真核双表达载体pIRES-hVEGF_（121）/hBMP-4的构建与鉴定
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摘要
目的构建含有人血管内皮生长因子(human Vascular endothelial growth factor,hVEGF121)和人骨形态发生蛋白-4(human Bone morphogenesia protein-4,hBMP-4)两基因的真核双表达质粒,并鉴定。
方法首先将hVEGF121通过定向克隆至真核双表达质粒pIRES一个多克隆位点中,命名为pIRES-hVEGF121。再将hBMP-4定向克隆至穿梭质粒pShuttle CMV上,构建pShuttle CMV-hBMP-4重组穿梭质粒,最后通过重组穿梭质粒将hBMP-4定向克隆至真核双表达质粒pIRES-hVEGF121上,完成真核双表达载体pIRES- hVEGF121/hBMP-4的构建,并作酶切鉴定。转染COS-7细胞,提取总RNA和总蛋白,分别进行RT-PCR检测其转录和Western blot检测其蛋白表达。
结果酶切证明pIRES-hVEGF121真核表达载体构建正确;酶切证明pShuttle CMV-hBMP-4穿梭质粒构建正确;酶切证明真核双表达载体pIRESh-VEGF121/hBMP-4构建正确;RT-PCR检测到hVEGF121和hBMP- 4基因转录,Western- blot检测hVEGF121和hBMP-4基因在COS-7细胞中的表达。
结论真核双表达载体pIRES-hVEGF121/hBMP-4成功构建,RT-PCR检测到hVEGF121和hBMP-4基因转录,Western-blot检测到hVEGF121和hBMP-4基因在COS-7细胞中的表达。hVEGF121和hBMP-4在COS-7细胞中成功表达,为进一步研究打下良好基础。
Objective To construct the eukaryotic coexpression vector encoding human vascular endothelial growth factor (VEGF121) and human bone morphogenesia protein-4 (hBMP-4), and detect the expression of the plasmid after COS-7 cell being transformed into.
Methods Firstly, VEGF121 was directly cloned into one of the multiple cloning sites of the eukaryotic expression plasmid-pIRES to construct pIRES-hVEGF121 . Secondly,hBMP-4 was directly cloned into pShuttle CMV to construct pShuttle CMV-hBMP-4, a new reconstructed shuttle plasmid. Carried with the instrument of pShuttle CMV-hBMP-4,hBMP-4 was cloned into pIRES-hVEGF121 to construct pIRES-hVEGF121/hBMP-4, which laterly were confirmed by restriction enzymolysis and then transformed into COS-7. Thirdly, the transcription of the RNA extracted from COS-7 were detected by RT-PCR and the expression of proteins extracted from COS-7 were detected by Western blot assay.
Results The eukaryotic expression plasmid-pIRES-hVEGF121 could be digested by enzyme, which can certificate its right construction; the shuttle plasmid-pShuttle CMV-hBMP-4 could be digested by enzyme, which can certificate its right construction; the eukaryotic expression plasmid-pIRES-hVEGF121/hBMP-4 could be digested by enzyme, which can certificate its right construction. The transcription of hVEGF121 and hBMP-4 gene could be detected by RT-PCR and the proteins expression of hBMP-4 and hVEGF121 genes could be confirmed by Western-blot assay.
Conclusion The construction of pIRES-hVEGF121/hBMP-4 was acheved; the transcription of hVEGF121 and hBMP-4 gene could be detected by RT-PCR and the proteins expression of hBMP-4 and hVEGF121 genes could be confirmed by Western-blot assay; the proteins expression of hBMP-4 and hVEGF121 genes could set a sound ground for the further researches.
方法首先将hVEGF121通过定向克隆至真核双表达质粒pIRES一个多克隆位点中,命名为pIRES-hVEGF121。再将hBMP-4定向克隆至穿梭质粒pShuttle CMV上,构建pShuttle CMV-hBMP-4重组穿梭质粒,最后通过重组穿梭质粒将hBMP-4定向克隆至真核双表达质粒pIRES-hVEGF121上,完成真核双表达载体pIRES- hVEGF121/hBMP-4的构建,并作酶切鉴定。转染COS-7细胞,提取总RNA和总蛋白,分别进行RT-PCR检测其转录和Western blot检测其蛋白表达。
结果酶切证明pIRES-hVEGF121真核表达载体构建正确;酶切证明pShuttle CMV-hBMP-4穿梭质粒构建正确;酶切证明真核双表达载体pIRESh-VEGF121/hBMP-4构建正确;RT-PCR检测到hVEGF121和hBMP- 4基因转录,Western- blot检测hVEGF121和hBMP-4基因在COS-7细胞中的表达。
结论真核双表达载体pIRES-hVEGF121/hBMP-4成功构建,RT-PCR检测到hVEGF121和hBMP-4基因转录,Western-blot检测到hVEGF121和hBMP-4基因在COS-7细胞中的表达。hVEGF121和hBMP-4在COS-7细胞中成功表达,为进一步研究打下良好基础。
Objective To construct the eukaryotic coexpression vector encoding human vascular endothelial growth factor (VEGF121) and human bone morphogenesia protein-4 (hBMP-4), and detect the expression of the plasmid after COS-7 cell being transformed into.
Methods Firstly, VEGF121 was directly cloned into one of the multiple cloning sites of the eukaryotic expression plasmid-pIRES to construct pIRES-hVEGF121 . Secondly,hBMP-4 was directly cloned into pShuttle CMV to construct pShuttle CMV-hBMP-4, a new reconstructed shuttle plasmid. Carried with the instrument of pShuttle CMV-hBMP-4,hBMP-4 was cloned into pIRES-hVEGF121 to construct pIRES-hVEGF121/hBMP-4, which laterly were confirmed by restriction enzymolysis and then transformed into COS-7. Thirdly, the transcription of the RNA extracted from COS-7 were detected by RT-PCR and the expression of proteins extracted from COS-7 were detected by Western blot assay.
Results The eukaryotic expression plasmid-pIRES-hVEGF121 could be digested by enzyme, which can certificate its right construction; the shuttle plasmid-pShuttle CMV-hBMP-4 could be digested by enzyme, which can certificate its right construction; the eukaryotic expression plasmid-pIRES-hVEGF121/hBMP-4 could be digested by enzyme, which can certificate its right construction. The transcription of hVEGF121 and hBMP-4 gene could be detected by RT-PCR and the proteins expression of hBMP-4 and hVEGF121 genes could be confirmed by Western-blot assay.
Conclusion The construction of pIRES-hVEGF121/hBMP-4 was acheved; the transcription of hVEGF121 and hBMP-4 gene could be detected by RT-PCR and the proteins expression of hBMP-4 and hVEGF121 genes could be confirmed by Western-blot assay; the proteins expression of hBMP-4 and hVEGF121 genes could set a sound ground for the further researches.
引文
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2. Park J, Lutz R, Felszeghy E, Wiltfang J, et al. The effect on bone regeneration of a liposomal vector to deliver BMP-2 gene to bone grafts in peri-implant bone defects. Biomaterials, 2007; 28(17): 2772 ~ 82.
3. Lieberman JR, Daluiski A, Stevenson S, et al. The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. J Bone Joint Surg Am, 1999; 81(7): 905 ~ 17.
4. Kirker-Head CA. Potential applications and delivery strategies for bone morphogenetic proteins. Adv Drug Deliv Rev, 2000; 43(1): 65 ~ 92.
5. Stuart DD, Kao GY, Allen TM, et al. A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1. Cancer Gene Ther, 2000; 7 (3): 466 ~ 75.
6. Peng H, Usas A, Olshanski A, et al. VEGF improves, whereas sFlt1 inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res, 2005; 20(11): 2017 ~ 27.
7. John S, Min B, Leo DG, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. PNAS, 2002; 99(15): 9656 ~ 61.
8. Hairong P, Vonda W, Arvydas U, et al. Synergistic enhancement of bone formation and healing by stem cell–expressed VEGF and bone morphogenetic protein-4. J. Clin. Invest, 2002; 110(6): 751 ~ 9.
9. Keith DK, Yan C, Kenneth MC, et al. Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation. Biochem Biophys Res Commun , 2003; 308 (3): 636 ~ 45.
10. Yan C, Kenneth MC, et al. In vivo new bone formation by direct transfer of adenoviral-mediated bone morphogenetic protein-4 gene. Biochem Biophys ResCommun , 2002; 298 (1): 121 ~ 7.
11. Bouletreau PJ, Warren SM, Spector JA, et al. Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: implications for fracture healing. Plast Reconstr Surg. 2002; 109(7): 2384 ~ 97.
12. Huang YC, Kaigler D, Rice KG, et al. Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration. J Bone Miner Res. 2005; 20(5): 848 ~ 57.
13. Deckers MM, Bezooijen RL, et al. Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology, 2002; 143(4): 1545 ~ 53.
14. Ferrara N, Davis-Smyth T, et al. The biology of vascular endothelial Growth factor. EndoerRev, 1997; 18(1): 4 ~ 25.
15. Nagy JA, Vasile E, Feng D, et al. Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med, 2002; 196(11): 1497 ~ 506.
16. Kroll J, Waltenberger J, et al. Regulation of the endothelial function and angiogenesis by vascular endotherlial growth factor(VEGF-A). ZKardiol, 2000; 19(3): 206 ~ 18.
17. Ikeda Y, Fukuda N, Wada M, et al. Development of angiogenic Cell and gene therapy by transplantation of umbilical cord blood With vascular endothelial growth factor gene. Hypertens Res, 2004; 27(2): 119 ~ 28.
18. Hiltunen MO, Ruuskanen M, Huuskonen J, et al. Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo. FASEB J, 2003; 17(9): 1147 ~ 9.
19. Shimasaki S, Zachow RJ, Li D, et al. A functional bone Morphogenetic protein system in the ovary. Proc Natl Acad Sci USA, 1999; 96(13): 7282 ~ 7.
20. Shore EM, Xu M, Shah PB, et al. The human bone morphogenetic protein
4(BMP-4) gene: molecular structure and transcriptional regulation. Calcif Tissue Int, 1998; 63(3): 221 ~ 229.
21. Pecina M, Jelic M, Martinovic S, et al. Articular cartilage repair: the role of bone morphogenetic proteins. Int Orthop, 2002; 26(3): 131 ~ 136.
22. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2,-4,and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005; 320(2): 269 ~ 276.
23. Roelen BA, Dijke P, et al. Controlling mesenchymal stem cell differentiation by TGFBeta family members. J Orthop Sci, 2003; 8(5): 740 ~ 748.
24. 汪炬, 孙奋勇, 戴云, 等. 由 BMP-4 基因转入 C2C12 细胞引起的成肌细胞到成骨细胞的表型转变. 中国病理生理杂志, 2004; 20(5): 719 ~ 723.
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