新型Tet-On反式激活因子对GDNF基因腺相关病毒载体表达调控的研究  

Regulation of AAV-mediated glial cell-line derived neurotrophic factor expression by using improved Tet-on trans-activator

作　　者：陈腾[1] 李新钢[1] 吴承远[1] 冈田尚己 小泽敬也[2] 

机构地区：[1]山东大学齐鲁医院神经外科,济南250012 [2]日本自治医科大学分子病态治疗研究中心遗传子治疗研究部

出　　处：《中华医学杂志》2005年第6期405-408,共4页National Medical Journal of China

摘　　要：目的　通过在胶质细胞源性神经营养因子(GDNF)基因腺相关病毒(AAV)表达载体中插入改良的Tet On调控因子,达到有目的调节GDNF的表达,避免基因过度表达或重组可能对宿主产生的危害。方法　首先在pAAV- GDNFflag前病毒质粒的hHG部分插入寡核苷酸序列,通过含相应酶切位点的引物,聚合酶链反应(PCR)扩增反式激活因子rtTA_2s S_2和四环素反应元件 (TRE),并插入寡核苷酸序列,构建pAAV- rtTA_2s -S_2- TRE -GDNFflag。与辅助质粒共转染HEK293细胞完成重组腺相关病毒(rAAV)载体的包装,氯化铯梯度超速离心分离病毒裂解液,半透膜提纯病毒载体,实时定量PCR(RQ- PCR)检测病毒效价。rAAV感染HEK293细胞,荧光显色和Western印迹法检测rtTA_2s S_2的体外调控;rAAV感染Wistar小鼠腓肠肌,酶联免疫吸附实验 (ELISA)检测GDNF基因表达的体内调控。结果　体外实验强力霉素阳性组Western印迹可见明显GDNF条带,阴性组未见;体内实验强力霉素阳性组GDNF在 2周内表达 (32 .6pg/ml±2 .6pg/ml),高于强力霉素阴性组 ( 10 .1pg/ml±2 .4pg/ml),差异有统计学意义。结论　Tet -On反式激活因子rtTA_2s S_2、TRE和GDNF基因可构建在同一AAV载体,通过调节诱导剂强力霉素的浓度,rtTA2s -S2在体内和体外均能有效调控下游目的基因GDNF的表达。Objective To control the expression of AAV mediated glial cell line derived neurotrophic factor (GDNF) gene purposely by incorporating novel Tet On trans activator rtTA2s S2, which prevents potential harms caused by over expression of recombinant target genes.Methods Oligonucleotide with specific monocloning sites was inserted into the hHG part of pAAV GDNFflag. Trans activator from pUHrT61 rtTA2s S2 and TRE from pTRE d2EGFP were amplified by PCR and inserted into pCRII TOPO respectively. Possible mutation was eliminated by sequencing. TRE and rtTA2s S2 were then inserted into the oligonucleotide of pAAV GDNFflag to form pAAV rtTA2s S2 TRE GDNFflag. pAAV rtTA2s S2 TRE d2EGFP was constructed by replacing the GDNFflag part with d2EGFP. The plasmids were digested by Xba I and compared with theoretic values. HEK 293 cells were cultured and co transfected with pAAV GDNFflag and helper plasmids pHLP19 and pAdeno5 so as to complete the package of recombinant adeno associated virus (rAAV) Crude virus lysate was purified by two sequential continuous CsCl gradient ultracentrifugation, dialyzed and condensed by millipore filter. The titer of rAAV was determined by real time quantitative PCR. Another HEK293 cells were cultured, transfected with rAVV, and then cultured in 2 kinds of culture fluid: with or without doxycyclin (Dox). Fluorescence microscopy was used to calculate the percentage of fluorescent cells so as to detect AAV rtTA2s S2 TRE d2EGFP, and Western blotting was used to detect the GDNF protein in the lysate of the HEK293 cells, thus testifying the regulatory function in vitro of rtTA2s S2. Twenty male Wistar mice were randomly divided into 2 groups: experiment group, fed with Dox and sucrose (Dox positive group), and control group, fed with only sucrose (Dox negative group). Two days after AAV rtTA2s S2 TRE GDNF was injected into the gastrocnemius muscles of the mice. Two weeks the mice were killed and their gastrocnemius muscles were taken out. ELISA was used to examine

关 键 词：GDNF基因 强力霉素 表达调控 反式激活因子 体内 阴性 TA 酶切位点 目的基因 腺相关病毒载体 

分 类 号：R741[医药卫生—神经病学与精神病学]