CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS SHUTTLE VECTOREXPRESSING NERVE GROWTH FACTOR β GENE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Ruofei ¹ , GAO Dalong ¹ , DANG Xiaoqian ² , WANG Kunzheng ² , YANG Leigang ¹ , TU Zhongmin. ¹

1 No.1 Department of Orthopedics, Xianyang Center Hospital, Xianyang Shaanxi, 712000, P.R.China;;
2No.1 Department of Orthopedics, the Second Affiliated Hospital of Medical College, Xi’an Jiaotong University.;

Corresponding author：

Keywords：

Nerve growth factor β; Adeno-associated virus vector; Shuttle vector; Spinal cord injury; Gene therapy

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【Abstract】 Objective To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nerve
growth factor β (NGF-β) gene. Methods By PCR amplification, the structural element of pAAV-multi ple cloning site
(MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA l igase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell l ine was cultured, and total RNA was extracted, then human NGF-β gene was obtained by RT-PCR. T-easy-NGF-β vector was constructed by cloning human NGF-β gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-β gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-β gene was obtained and identified by RT-PCR, digestion, and DNA sequencing. Results The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMVEGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-β was digested; DNA sequencing result of T-easy-NGF-β was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-β was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-β showed that sequences were completely correct. Conclusion The AAV shuttle vector pAAV-U6 /CMV-NGF-β is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.

Citation： LI Ruofei,GAO Dalong,DANG Xiaoqian,WANG Kunzheng,YANG Leigang,TU Zhongmin.. CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS SHUTTLE VECTOREXPRESSING NERVE GROWTH FACTOR β GENE. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 172-176. doi: Copy

1.	Demir O, Singh S, Klimaschewski L, et al. From birth till death: neurogenesis, cell cycle, and neurodegeneration. Anat Rec (Hoboken), 2009, 292(12): 1953-1961.
2.	Lee SI, Kim BG, Hwang DH, et al. Overexpression of Bcl-XL in human neural stem cells promotes graft survival and functional recovery following transplantation in spinal cord injury. J Neurosci Res, 2009, 87(14): 3186-3197.
3.	Lee SH, Chung YN, Kim YH, et al. Effects of human neural stem cell transplantation in canine spinal cord hemisection. Neurol Res, 2009, 31(9): 996-1002.
4.	Bethea CL, Reddy AP, Pedersen D, et al. Expression profile of differentiating serotonin neurons derived from rhesus embryonic stem cells and comparison to adult serotonin neurons. Gene Expr Patterns, 2009, 9(2): 94-108.
5.	Chigr F, Rachidi F, Segura S, et al. Neurogenesis inhibition in the dorsal vagal complex by chronic immobilization stress in the adult rat. Neuroscience, 2009, 158(2): 524-536.
6.	Tan YX, Yu JX, Wang DF. Effects of NGF pretreatment on cerebral injury in gerbils. J Nanjing Medical University, 2009, 23(4): 265-269.
7.	范广明, 张文彬, 张赛, 等. 神经生长因子联合神经干细胞移植治疗大鼠脊髓损伤. 中国组织工程研究与临床康复, 2010, 14(14): 2572-2578.
8.	Adriaenssens E, Vanhecke E, Saule P, et al. Nerve growth factor is a potential therapeutic target in breast cancer. Cancer Res, 2008, 68(2): 346-351.
9.	花柱明, 李真, 罗东兰. 神经生长因子在胰腺癌中的表达与癌细胞嗜神经侵袭和疼痛的关系. 南方医科大学学报, 2006, 26(8): 1251-1253.
10.	孟令新, 丁兆军, 陈希平, 等. 神经生长因子及其受体在人胰腺导管癌组织中的表达. 中华内科杂志, 2009, 48(7): 562-565.
11.	王选, 雷晓婷, 丁新生. 脊髓损伤后几类促神经修复分子作用机制研究进展. 神经损伤与功能重建, 2009, 4(2): 131-132.
12.	Diensthuber M, Lenarz T, Stöver T. Neurotrophic factor expression in vestibular schwannoma. An overview. Laryngorhinootologie, 2006, 85(10): 731-737.
13.	Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection and neural repair. Annu Rev Neurosci, 2001, 24: 1217-1281.
14.	Chiaretti A, Genovese O, Riccardi R, et al. Intraventricular nerve growth factor infusion: a possible treatment for neurological deficits following hypoxic-ischemic brain injury in infants. J Neurosci Res, 2005, 27(7): 741-746.
15.	Williams BJ, Eriksdotter, Jonhagen M, et al. Nerve growth factor in treatment and pathogenesis of Alzheimer’s disease. Prog Neurobiol, 2006, 80(3): 114-128.
16.	Brown A, Ricci MJ, Weaver LC. NGF message and protein distribution in the injured rat spinal cord. Exp Neurol, 2004, 188(1): 115-127.
17.	Brown A, Ricci MJ, Weaver LC. NGF mRNA is expressed in the dorsal root ganglia after spinal cord injury in the rat. Exp Neurol, 2007, 205(1): 283-286.
18.	Yamasaki K, Setoguchi T, Takenouchi T, et al. Stem cell factor prevents neuronal cell apoptosis after acute spinal cord injury. Spine (Phila Pa 1976), 2009, 34 (4): 323-327 .
19.	Foust KD, Nurre E, Montgomery CL, et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009, 27(1): 59-65.
20.	Li C, Bowles DE, van Dyke T, et al. Adeno-associated virus vectors: potential applications for cancer gene therapy. Cancer Gene Ther, 2005, 12 (12): 913-925.
21.	Blouin V, Brument N, Toublanc E , et al. Improving rAAV production and purification: towards the definition of ascaleable process. J Gene Med, 2004, 6 Suppl 1: 5223-5228.

1. Demir O, Singh S, Klimaschewski L, et al. From birth till death: neurogenesis, cell cycle, and neurodegeneration. Anat Rec (Hoboken), 2009, 292(12): 1953-1961.
2. Lee SI, Kim BG, Hwang DH, et al. Overexpression of Bcl-XL in human neural stem cells promotes graft survival and functional recovery following transplantation in spinal cord injury. J Neurosci Res, 2009, 87(14): 3186-3197.
3. Lee SH, Chung YN, Kim YH, et al. Effects of human neural stem cell transplantation in canine spinal cord hemisection. Neurol Res, 2009, 31(9): 996-1002.
4. Bethea CL, Reddy AP, Pedersen D, et al. Expression profile of differentiating serotonin neurons derived from rhesus embryonic stem cells and comparison to adult serotonin neurons. Gene Expr Patterns, 2009, 9(2): 94-108.
5. Chigr F, Rachidi F, Segura S, et al. Neurogenesis inhibition in the dorsal vagal complex by chronic immobilization stress in the adult rat. Neuroscience, 2009, 158(2): 524-536.
6. Tan YX, Yu JX, Wang DF. Effects of NGF pretreatment on cerebral injury in gerbils. J Nanjing Medical University, 2009, 23(4): 265-269.
7. 范广明, 张文彬, 张赛, 等. 神经生长因子联合神经干细胞移植治疗大鼠脊髓损伤. 中国组织工程研究与临床康复, 2010, 14(14): 2572-2578.
8. Adriaenssens E, Vanhecke E, Saule P, et al. Nerve growth factor is a potential therapeutic target in breast cancer. Cancer Res, 2008, 68(2): 346-351.
9. 花柱明, 李真, 罗东兰. 神经生长因子在胰腺癌中的表达与癌细胞嗜神经侵袭和疼痛的关系. 南方医科大学学报, 2006, 26(8): 1251-1253.
10. 孟令新, 丁兆军, 陈希平, 等. 神经生长因子及其受体在人胰腺导管癌组织中的表达. 中华内科杂志, 2009, 48(7): 562-565.
11. 王选, 雷晓婷, 丁新生. 脊髓损伤后几类促神经修复分子作用机制研究进展. 神经损伤与功能重建, 2009, 4(2): 131-132.
12. Diensthuber M, Lenarz T, Stöver T. Neurotrophic factor expression in vestibular schwannoma. An overview. Laryngorhinootologie, 2006, 85(10): 731-737.
13. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection and neural repair. Annu Rev Neurosci, 2001, 24: 1217-1281.
14. Chiaretti A, Genovese O, Riccardi R, et al. Intraventricular nerve growth factor infusion: a possible treatment for neurological deficits following hypoxic-ischemic brain injury in infants. J Neurosci Res, 2005, 27(7): 741-746.
15. Williams BJ, Eriksdotter, Jonhagen M, et al. Nerve growth factor in treatment and pathogenesis of Alzheimer’s disease. Prog Neurobiol, 2006, 80(3): 114-128.
16. Brown A, Ricci MJ, Weaver LC. NGF message and protein distribution in the injured rat spinal cord. Exp Neurol, 2004, 188(1): 115-127.
17. Brown A, Ricci MJ, Weaver LC. NGF mRNA is expressed in the dorsal root ganglia after spinal cord injury in the rat. Exp Neurol, 2007, 205(1): 283-286.
18. Yamasaki K, Setoguchi T, Takenouchi T, et al. Stem cell factor prevents neuronal cell apoptosis after acute spinal cord injury. Spine (Phila Pa 1976), 2009, 34 (4): 323-327 .
19. Foust KD, Nurre E, Montgomery CL, et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009, 27(1): 59-65.
20. Li C, Bowles DE, van Dyke T, et al. Adeno-associated virus vectors: potential applications for cancer gene therapy. Cancer Gene Ther, 2005, 12 (12): 913-925.
21. Blouin V, Brument N, Toublanc E , et al. Improving rAAV production and purification: towards the definition of ascaleable process. J Gene Med, 2004, 6 Suppl 1: 5223-5228.

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS SHUTTLE VECTOREXPRESSING NERVE GROWTH FACTOR β GENE

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