大鼠VGLUT2基因shRNA慢病毒载体的构建和鉴定  

The construction and identifying of lentiviral vectors that express shRNA targeting VGLUT2 gene of rats

作　　者：姜俊[1] 高凡 葛顺楠 李志红 唐君 李金莲 

机构地区：[1]第四军医大学航空医学系2009级学员 [2]口腔医学系2008级学员 [3]人体解剖学教研室暨梁銶琚脑研究中心,西安710032

出　　处：《神经解剖学杂志》2011年第6期597-603,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81070900)

摘　　要：目的:构建可以表达针对2型囊泡膜谷氨酸转运体(VGLUT2)shRNA的慢病毒载体,特异性下调中枢神经系统内VGLUT2的表达,从而为研究VGLUT1和VGLUT2的功能差异提供有力的工具。方法:首先在体外合成四对互补并靶向作用于编码大鼠VGLUT2序列四个位点的特异性短发卡RNA(short hairpin RNA)序列和一个阴性对照的寡核苷酸,克隆到pGCSIL-GFP质粒(经Age I和EcoRI双酶切)载体内,经酶切及测序鉴定正确后,将该质粒与辅助质粒pHelper1.0以及pHelper2.0共转染T293细胞,包装得到病毒颗粒。各组病毒载体转染原代培养的大鼠皮层神经元后,运用免疫荧光和Western Blot检测各组蛋白的表达水平。结果:pGCSIL-GFP质粒克隆得到的VGLUT2 shRNA序列正确,包装得到的病毒可以有效转染原代培养的皮层神经元,Western Blot检测结果显示:VGLUT2 shRNA-1与VGLUT2 shRNA-2病毒组的VGLUT2表达水平与空白组相比有显著性差异(P<0.05),分别为空白组的63.9±5.82%,73.8±3.18%;VGLUT2 shRNA-3与VGLUT2 shRNA-4病毒组的VGLUT2表达水平与空白组相比同样有显著性差异(P<0.05),分别为空白组的39.1±1.99%,35.1±1.72%;VGLUT2shRNA-1与VGLUT2 shRNA-2病毒组相比无显著性差异(P>0.05);但VGLUT2 shRNA-3与VGLUT2 shRNA-4病毒组与VGLUT2 shRNA-1和VGLUT2 shRNA-2病毒组相比有显著性差异(P<0.05)。表明VGLUT2 shRNA-3与VGLUT2 shRNA-4组病毒能较为高效的下调培养的皮层神经元VGLUT2的表达,下调效率超过60%。结论:本研究成功构建出表达针对大鼠VGLUT2基因shRNA的慢病毒载体,并可有效下调VGLUT2的表达,为进一步研究VGLUT2的功能及其与VGLUT1之间的功能差异提供了有力的工具。Objective： To construct the lentiviral vectors which express the shRNA targeting the vesicular glutamate transporter 2（VGLUT2）and specifically down-regulate the expression of VGLUT2 in the central nervous system,for obtaining the powerful tool to explore the functional difference of the VGLUT1 and VGLUT2.Methods： Four pairs of specific shRNA sequences which target the four sites of VGLUT2 coding sequences and one pair of negative control oligonucleotide sequence were cloned into the pGCSIL-GFP plasmid（restricted by Age I and EcoRI）.After identified by restriction and sequencing,the pGCSIL-GFP plasmid and two helper plasmids pHelper1.0 and pHelper2.0 were co-transfected into the T293 cells,and the viral particles were packaged.The primary cultured rats cortical neurons were transfected by the groups of lentiviral vectors,and the VGLUT2 expressional levels were examined by immonofluorescence and Western Blot.Results： The sequences of the VGLUT2 shRNA cloned by pGCSIL-GFP plasmid were correct and the packaged lentiviral vectors can be effectively transfected into the primary cultured cortical neurons.The Western Blot examination showed： The VGLUT2 expressional level for VGLUT2 shRNA-1 and VGLUT2 shRNA-2 group was significant difference comparing with that for vehicle group（P〈0.05）,reaching to 63.9±5.82% and 73.8±3.18% of vehicle group respectively;the VGLUT2 expressional level for VGLUT2 shRNA-3 and VGLUT2 shRNA-4 group was significant difference comparing with that for vehicle group as well（P〈0.05）,reaching to 39.1±1.99% and 35.1±1.72% of vehicle group respectively;there is no significant difference between the VGLUT2 shRNA-1 and VGLUT2 shRNA-2 group（P〈0.05）;while the VGLUT2 expressional level for VGLUT2 shRNA-3 and VGLUT2 shRNA-4 group was significant difference comparing with that for VGLUT2 shRNA-1 and VGLUT2 shRNA-2 group（P〈0.05）.The results showed that the VGLUT2 shRNA-3 and VGLUT2 shRNA-4 can better effectively down-regulate the expression of VGLUT2 by primary cultu

关 键 词：VGLUT2 RNA干扰 慢病毒载体 原代培养神经元 大鼠 

分 类 号：Q78[生物学—分子生物学]