• 1 四川大學(xué)華西醫(yī)院骨科（成都，610041）;;
  • 2 寧夏醫(yī)科大學(xué)附屬總醫(yī)院脊柱骨科;

【摘 要】 目的 構(gòu)建NEP1-40（Nogo extra cellular peptide residues 1-40）基因慢病毒表達(dá)載體，為后續(xù)轉(zhuǎn)染目
的細(xì)胞奠定基礎(chǔ)，并實(shí)現(xiàn)在細(xì)胞中高效、穩(wěn)定表達(dá)。 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(qū)片段。將目的基因與酶切線性化的載體pGC-FU 進(jìn)行定向連接，其產(chǎn)物轉(zhuǎn)化細(xì)菌感受態(tài)細(xì)胞。對長
出的克隆先進(jìn)行菌落PCR 鑒定，再對PCR 鑒定陽性的克隆進(jìn)行測序和比對分析，比對正確的克隆即為構(gòu)建成功的目的質(zhì)
粒。將構(gòu)建成功的目的質(zhì)粒和兩種輔助包裝質(zhì)粒共轉(zhuǎn)染293T 細(xì)胞，包裝成慢病毒，熒光顯微鏡下觀察慢病毒轉(zhuǎn)染293T
細(xì)胞后熒光表達(dá)情況，采用Western blot 檢測NEP1-40 及綠色熒光蛋白融合蛋白表達(dá)情況。 結(jié)果 PCR 產(chǎn)物經(jīng)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(zhuǎn)染質(zhì)粒連接構(gòu)建正確；熒光表達(dá)檢測顯示293T 細(xì)胞中產(chǎn)
生慢病毒顆粒；Western blot 顯示NEP1-40 在細(xì)胞內(nèi)穩(wěn)定表達(dá)。 結(jié)論 成功構(gòu)建NEP1-40 基因慢病毒表達(dá)載體，為后續(xù)
轉(zhuǎn)染目的細(xì)胞后從分子水平探討NEP1-40 基因功能奠定了實(shí)驗(yàn)基礎(chǔ)。

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(gòu)建及鑒定. 中國修復(fù)重建外科雜志, 2012, 26(2): 177-181. doi: 復(fù)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(dá)和純化. 中國修復(fù)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(dá)腺病毒載體的構(gòu)建及體外轉(zhuǎn)染大鼠BMSCs的初步實(shí)驗(yàn)研究. 中國修復(fù)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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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(dá)和純化. 中國修復(fù)重建外科雜志, 2006, 20(1): 9-12.
  15. 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. 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. 17. 薛靜, 彭江, 張莉, 等. 綠色熒光蛋白和Nel1型蛋白基因共表達(dá)腺病毒載體的構(gòu)建及體外轉(zhuǎn)染大鼠BMSCs的初步實(shí)驗(yàn)研究. 中國修復(fù)重建外科雜志, 2010, 24(5): 606-612.
  18. 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. 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. 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.