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9.1×10~6 cfu/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。
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-SHRE-NT3. After purification and concentration, the retrovirus titer reached 9. 1 X 106 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.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2016年第2期190-194,共5页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.81301041)
陕西省青年科技新星项目(No.2015KJXX-43)
陕西省优势学科建设经费资助[No.陕教办(2014)3-1001]
陕西省教育厅专项科研计划项目(No.2013JK0756
14JK1614)
青海省应用基础研究资助项目(No.2013-Z-727)~~
作者简介
通讯作者：徐曦．E—mail：xmux@qq．com
