CONSTRUCTION AND IDENTIFICATION OF Nogo EXTRA CELLULAR PEPTIDE RESIDUES 1-40 GENELENTIVIRAL VECTOR_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUAN Haifeng ^1,2 , SONG Yueming ¹ , LIU Hao ¹ , ZHOU Chunguang ¹ , KONG Qingquan ¹ , LIU Liming ¹ , GONG Quan ¹

1Department of Orthopedics， West China Hospital， Sichuan University， Chengdu Sichuan, 610041, P.R.China;;
2Department of Spinal Surgery, Affiliated General Hospital, Ningxia Medical University.;

Corresponding author：

Keywords：

Nogo extra cellular peptide residues 1-40; Nerve regeneration; Lentivirus vector; 293T cells

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【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40
(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment of
NEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.

Citation： YUAN Haifeng,SONG Yueming,LIU Hao,ZHOU Chunguang,KONG Qingquan,LIU Liming,GONG Quan. CONSTRUCTION AND IDENTIFICATION OF Nogo EXTRA CELLULAR PEPTIDE RESIDUES 1-40 GENELENTIVIRAL VECTOR. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 177-181. doi: Copy

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.	宫福良, 王坤正, 余鹏博, 等. NEP1-40基因克隆及蛋白的原核表达和纯化. 中国修复重建外科杂志, 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.	薛静, 彭江, 张莉, 等. 绿色荧光蛋白和Nel1型蛋白基因共表达腺病毒载体的构建及体外转染大鼠BMSCs的初步实验研究. 中国修复重建外科杂志, 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. 宫福良, 王坤正, 余鹏博, 等. NEP1-40基因克隆及蛋白的原核表达和纯化. 中国修复重建外科杂志, 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. 薛静, 彭江, 张莉, 等. 绿色荧光蛋白和Nel1型蛋白基因共表达腺病毒载体的构建及体外转染大鼠BMSCs的初步实验研究. 中国修复重建外科杂志, 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.

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION AND IDENTIFICATION OF Nogo EXTRA CELLULAR PEPTIDE RESIDUES 1-40 GENELENTIVIRAL VECTOR

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