西安交通大学学报（医学版）Issue(2)：190-194,5.DOI:10.7652/jdyxb201602009
缺氧调控表达的 NT-3逆转录病毒载体的构建及鉴定
Construction and identification of the recombinant retroviral vector to carry out hypoxia-regulated expression of neurotrophin-3
摘要
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 48 h 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 × 10 6 cfu/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.关键词
逆转录病毒载体/缺氧调控表达/神经营养因子-3Key words
retroviral vector/hypoxia-regulated expression/neurotrophin-3 (NT-3)分类
医药卫生引用本文复制引用
张军峰,史利利,张力,杨蓬勃,张建水,刘勇,祁存芳,徐曦..缺氧调控表达的 NT-3逆转录病毒载体的构建及鉴定[J].西安交通大学学报（医学版）,2016,(2):190-194,5.基金项目
国家自然科学基金资助项目(No.81301041) （No.81301041）
陕西省青年科技新星项目(No.2015KJXX-43) （No.2015KJXX-43）
陕西省优势学科建设经费资助[No.陕教办(2014)3-1001] （2014）
陕西省教育厅专项科研计划项目(No.2013JK0756、14JK1614) （No.2013JK0756、14JK1614）
青海省应用基础研究资助项目(No.2013-Z-727) Supported by the National Natural Science Foundation of China (No.81301041),Scientific Research Program Funded by the Depart-ment of Science and Technology of Shaanxi Province (No.2015KJXX-43),the Leading Disciplines Development Government Founda-tion of Shaanxi (No.[2014]3-1001),the Scientific Research Program Funded by Shaanxi Provincial Education Department (No.2013JK0756,14JK1614),and Basic Application Research Program Funded by Qinghai Science & Technology Department (No.2013-Z-727) （No.2013-Z-727）
