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- 2016
缺氧调控表达的NT-3逆转录病毒载体的构建及鉴定
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Abstract:
摘要：目的 构建与鉴定含有5个拷贝缺氧反应元件(five copies of hypoxia responsive elements, 5HRE)的缺氧调控表达的神经营养因子-3(neurotrophin-3, NT-3)的逆转录病毒载体。方法 用PCR、酶切和连接等分子克隆技术将5HRE和人来源NT-3片段克隆入逆转录病毒载体pLNCX中，构建重组逆转录病毒穿梭质粒pLNCX-5HRE-SV40-NT3-IRES-EGFP。经PT67包装细胞包装、高速离心纯化和浓缩等方法，获得逆转录病毒RV-5HRE-NT3；用逆转录病毒感染NIH 3T3细胞48h后，流式细胞仪检测并计算出病毒滴度。 结果 成功构建了重组逆转录病毒载体质粒pLNCX-5HRE-SV40-NT3-IRES-EGFP，并获得相应的逆转录病毒RV-5HRE-NT3，经过高速离心纯化和浓缩后，其滴度可达9.1×106cfu/mL。结论 成功构建了缺氧调控表达的NT-3逆转录病毒载体，为研究外源基因的缺氧调控特异性表达奠定了工作基础。
ABSTRACT: Objective To construct and identify the recombinant retroviral vector containing five copies of hypoxia responsive elements (5HRE) and neurotrophin-3 (NT-3). Methods Using PCR, enzyme digestion and DNA ligase, 5HRE and human derived NT-3 were cloned into the retroviral vector plasmid (pLNCX) to construct the recombinant retroviral vector plasmid pLNCX-5HRE-SV40-NT3-IRES-EGFP. The retrovirus RV-5HRE-NT3 was packaged in the PT67 cells, and then it was purified and concentrated by high-speed centrifugation. After infected for 48h with the concentrated retrovirus, the number of the EGFP positive cells in the NIH 3T3 cells was counted by fluorescence activated cells and sorted to calculate the retrovirus titer. Results The retroviral vector plasmid, pLNCX-5HRE-SV40-NT3-IRES-EGFP, was successfully constructed, and the retrovirus was packaged and defined as RV-5HRE-NT3. After purification and concentration, the retrovirus titer reached 9.1×106cfu/mL. Conclusion The recombinant retroviral vector which carried out hypoxia-regulated expression of NT-3 was successfully constructed. It may provide basis for studies on hypoxia-regulated expression of the exogenous genes
| [1] | SHEN F, FAN Y, SU H, et al. Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice[J]. Gene Ther, 2008, 15(1):30-39. |
| [2] | SHITABA T, GIACCIA AJ, BROWN JM. Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy[J]. Neoplasia, 2002, 4(1):40-48. |
| [3] | NAYLOR LH. Reporter gene technology: the future looks bright[J]. Biochem Pharmacol, 1999, 58(5):749-757. |
| [4] | CAI J, HUA F, YUAN L, et al. Potential therapeutic effects of neurotrophins for acute and chronic neurological diseases[J]. Biomed Res Int, 2014, 2014:601084. |
| [5] | KIM HA, LIM S, MOON HH, et al. Hypoxia-inducible vascular endothelial growth factor gene therapy using the oxygen-dependent degradation domain in myocardial ischemia[J]. Pharm Res, 2010, 27(10):2075-2084. |
| [6] | BRANDL M. Liposomes as drug carriers: a technological approach[J]. Biotechnol Annu Rev, 2001, 7:59-85. |
| [7] | WEINSTEIN JN. Liposomes as drug carriers in cancer therapy[J]. Cancer Treat Rep, 1984, 68(1):127-135. |
| [8] | BARTEAU B, CHEVRE R, LETROUBONNEVAL E, et al. Physicochemical parameters of non-viral vectors that govern transfection efficiency[J]. Curr Gene Ther, 2008, 8(5):313-323. |
| [9] | CHEN JL, WANG H, GAO JQ, et al. Liposomes modified with polycation used for gene delivery: preparation, characterization and transfection <strong>i<em>n vitro</em></strong>[J]. Int J Pharm, 2007, 343(1-2):255-261. |
| [10] | 马进财，刘吉勇，刘绍玲. 两种重组逆转录病毒浓缩方法的比较[J]. 山东医药, 2006, 46(7):20-21. |
| [11] | SHI Q, ZHANG P, ZHANG J, et al. Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice[J]. Neurosci Lett, 2009, 465(3):220-225. |
| [12] | MILLER AD. Retroviral vector production[J]. Curr Protoc Hum Genet, 2014, 80:12-15. |
| [13] | LIPPINCOTT-SCHWARTZ J, PATTERSON GH. Development and use of fluorescent protein markers in living cells[J]. Science, 2003, 300(5616):87-91. |
