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

作　　者：张军峰[1] 史利利[1] 张力[1] 杨蓬勃[2] 张建水[2] 刘勇 祁存芳[3] 徐曦[1] 

机构地区：[1]西安医学院人体解剖学教研室,陕西西安710021 [2]西安交通大学医学部神经生物学研究所,陕西西安710061 [3]青海大学医学院基础医学部解剖学教研室,青海西宁810001

出　　处：《西安交通大学学报（医学版）》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)~~

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.

关 键 词：逆转录病毒载体 缺氧调控表达 神经营养因子-3 

分 类 号：R338[医药卫生—人体生理学] R741.02[医药卫生—基础医学]