NEP1-40 基因慢病毒載體構建及鑒定_《中國修復重建外科雜志》

作者：

袁海峰 ^1,2 , 宋躍明 ¹ , 劉浩 ¹ , 周春光 ¹ , 孔清泉 ¹ , 劉立岷 ¹ , 龔全 ¹

1 四川大學華西醫院骨科（成都，610041）;;
2 寧夏醫科大學附屬總醫院脊柱骨科;

關鍵詞：

NEP1-40 神經再生慢病毒載體 293T 細胞

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摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

【摘要】目的構建NEP1-40（Nogo extra cellular peptide residues 1-40）基因慢病毒表達載體，為后續轉染目
的細胞奠定基礎，并實現在細胞中高效、穩定表達。方法從含有 NEP1-40 基因的 cDNA 文庫中，利用PCR 方法釣取
NEP1-40 基因編碼區片段。將目的基因與酶切線性化的載體pGC-FU 進行定向連接，其產物轉化細菌感受態細胞。對長
出的克隆先進行菌落PCR 鑒定，再對PCR 鑒定陽性的克隆進行測序和比對分析，比對正確的克隆即為構建成功的目的質
粒。將構建成功的目的質粒和兩種輔助包裝質粒共轉染293T 細胞，包裝成慢病毒，熒光顯微鏡下觀察慢病毒轉染293T
細胞后熒光表達情況，采用Western blot 檢測NEP1-40 及綠色熒光蛋白融合蛋白表達情況。結果 PCR 產物經電泳分
析表明成功獲取NEP1-40 基因cDNA 克隆，測序提示慢病毒轉染質粒連接構建正確；熒光表達檢測顯示293T 細胞中產
生慢病毒顆粒；Western blot 顯示NEP1-40 在細胞內穩定表達。結論成功構建NEP1-40 基因慢病毒表達載體，為后續
轉染目的細胞后從分子水平探討NEP1-40 基因功能奠定了實驗基礎。

引用本文： 袁海峰 ,宋躍明,劉浩,周春光,孔清泉,劉立岷,龔全. NEP1-40 基因慢病毒載體構建及鑒定. 中國修復重建外科雜志, 2012, 26(2): 177-181. doi: 復制

1.	Williams G, Wood A, Williams EJ, et al. Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists. J Biol Chem, 2008, 283(24): 16641-16645.
2.	Wu J, Yang H, Qiu Z, et al. Effect of combined treatment with methylprednisolone and Nogo-A monoclonal antibody after rat spinal cord injury. J Int Med Res, 2010, 38(2): 570-582.
3.	Huebner EA, Kim BG, Duffy PJ, et al. A multi-domain fragment of Nogo-A protein is a potent inhibitor of cortical axon regeneration via Nogo receptor 1. J Biol Chem, 2011, 286(20): 18026-18036.
4.	Vasudevan SV, Schulz J, Zhou C, et al. Protein folding at the membrane interface, the structure of Nogo-66 requires interactions with a phosphocholine surface. Proc Natl Acad Sci U S A, 2010, 107(15): 6847-6851.
5.	Dupuis L, Pehar M, Cassina P, et al. Nogo receptor antagonizes p75NTR-dependent motor neuron death. Proc Natl Acad Sci U S A, 2008, 105(2): 740-745.
6.	Cao Y, Shumsky JS, Sabol MA, et al. Nogo-66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat. Neurorehabil Neural Repair, 2008 , 22(3): 262-278.
7.	Fang PC, Barbay S, Plautz EJ, et al. Combination of NEP 1-40 treatment and motor training enhances behavioral recovery after a focal cortical infarct in rats. Stroke, 2010, 41(3): 544-549.
8.	Hoffmann A, Bredno J, Wendland M, et al. High and low molecular weight fluorescein isothiocyanate (FITC)-dextrans to assess blood-brain barrier disruption: technical considerations. Transl Stroke Res, 2011, 2(1): 106-111.
9.	Luo Y, Wu X, Liu S, et al. Reactivation of visual cortical plasticity by NEP1-40 from early monocular deprivation in adult rats. Neurosci Lett, 2011, 494(3): 196-201.
10.	Mingorance A, Solé M, Munetón V, et al. Regeneration of lesioned entorhino-hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo-66/NgR signaling. FASEB J, 2006 , 20(3): 491-493.
11.	GrandPré T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature, 2000, 403(6768): 439-444.
12.	Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci, 2003, 23(10): 4219-4227.
13.	Gou X, Wang Q, Yang Q, et al. TAT-NEP1-40 as a novel therapeutic candidate for axonal regeneration and functional recovery after stroke. J Drug Target, 2011, 19(2): 86-95.
14.	宮福良, 王坤正, 余鵬博, 等. NEP1-40基因克隆及蛋白的原核表達和純化. 中國修復重建外科雜志, 2006, 20(1): 9-12.
15.	Joshi A, Garg H, Ablan S, et al. Targeting the HIV entry, assembly and release pathways for anti-HIV gene therapy. Virology, 2011, 415(2): 95-106.
16.	Hu B, Tai A, Wang P. Immunization delivered by lentiviral vectors for cancer and infectious diseases. Immunol Rev, 2011, 239(1): 45-61.
17.	薛靜, 彭江, 張莉, 等. 綠色熒光蛋白和Nel1型蛋白基因共表達腺病毒載體的構建及體外轉染大鼠BMSCs的初步實驗研究. 中國修復重建外科雜志, 2010, 24(5): 606-612.
18.	Nakashima H, Kaur B, Chiocca EA. Directing systemic oncolytic viral delivery to tumors via carrier cells. Cytokine Growth Factor Rev, 2010, 21(2-3): 119-126.
19.	Bertram CM, Hawes SM, Egli S, et al. Effective adenovirus-mediated gene transfer into neural stem cells derived from human embryonic stem cells. Stem Cells Dev, 2010 , 19(4): 569-578.
20.	Yang B, Sun HY, Chen WH, et al. Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation, and affects the cell cycle in LNCaP and PC3 cancer cell lines. Asian J Androl, 2010, 12(2): 196-202.

1. Williams G, Wood A, Williams EJ, et al. Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists. J Biol Chem, 2008, 283(24): 16641-16645.
2. Wu J, Yang H, Qiu Z, et al. Effect of combined treatment with methylprednisolone and Nogo-A monoclonal antibody after rat spinal cord injury. J Int Med Res, 2010, 38(2): 570-582.
3. Huebner EA, Kim BG, Duffy PJ, et al. A multi-domain fragment of Nogo-A protein is a potent inhibitor of cortical axon regeneration via Nogo receptor 1. J Biol Chem, 2011, 286(20): 18026-18036.
4. Vasudevan SV, Schulz J, Zhou C, et al. Protein folding at the membrane interface, the structure of Nogo-66 requires interactions with a phosphocholine surface. Proc Natl Acad Sci U S A, 2010, 107(15): 6847-6851.
5. Dupuis L, Pehar M, Cassina P, et al. Nogo receptor antagonizes p75NTR-dependent motor neuron death. Proc Natl Acad Sci U S A, 2008, 105(2): 740-745.
6. Cao Y, Shumsky JS, Sabol MA, et al. Nogo-66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat. Neurorehabil Neural Repair, 2008 , 22(3): 262-278.
7. Fang PC, Barbay S, Plautz EJ, et al. Combination of NEP 1-40 treatment and motor training enhances behavioral recovery after a focal cortical infarct in rats. Stroke, 2010, 41(3): 544-549.
8. Hoffmann A, Bredno J, Wendland M, et al. High and low molecular weight fluorescein isothiocyanate (FITC)-dextrans to assess blood-brain barrier disruption: technical considerations. Transl Stroke Res, 2011, 2(1): 106-111.
9. Luo Y, Wu X, Liu S, et al. Reactivation of visual cortical plasticity by NEP1-40 from early monocular deprivation in adult rats. Neurosci Lett, 2011, 494(3): 196-201.
10. Mingorance A, Solé M, Munetón V, et al. Regeneration of lesioned entorhino-hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo-66/NgR signaling. FASEB J, 2006 , 20(3): 491-493.
11. GrandPré T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature, 2000, 403(6768): 439-444.
12. Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci, 2003, 23(10): 4219-4227.
13. Gou X, Wang Q, Yang Q, et al. TAT-NEP1-40 as a novel therapeutic candidate for axonal regeneration and functional recovery after stroke. J Drug Target, 2011, 19(2): 86-95.
14. 宮福良, 王坤正, 余鵬博, 等. NEP1-40基因克隆及蛋白的原核表達和純化. 中國修復重建外科雜志, 2006, 20(1): 9-12.
15. Joshi A, Garg H, Ablan S, et al. Targeting the HIV entry, assembly and release pathways for anti-HIV gene therapy. Virology, 2011, 415(2): 95-106.
16. Hu B, Tai A, Wang P. Immunization delivered by lentiviral vectors for cancer and infectious diseases. Immunol Rev, 2011, 239(1): 45-61.
17. 薛靜, 彭江, 張莉, 等. 綠色熒光蛋白和Nel1型蛋白基因共表達腺病毒載體的構建及體外轉染大鼠BMSCs的初步實驗研究. 中國修復重建外科雜志, 2010, 24(5): 606-612.
18. Nakashima H, Kaur B, Chiocca EA. Directing systemic oncolytic viral delivery to tumors via carrier cells. Cytokine Growth Factor Rev, 2010, 21(2-3): 119-126.
19. Bertram CM, Hawes SM, Egli S, et al. Effective adenovirus-mediated gene transfer into neural stem cells derived from human embryonic stem cells. Stem Cells Dev, 2010 , 19(4): 569-578.
20. Yang B, Sun HY, Chen WH, et al. Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation, and affects the cell cycle in LNCaP and PC3 cancer cell lines. Asian J Androl, 2010, 12(2): 196-202.

《中國修復重建外科雜志》

NEP1-40 基因慢病毒載體構建及鑒定

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《中國修復重建外科雜志》

NEP1-40 基因慢病毒載體構建及鑒定

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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