慢病毒介导长链非编码RNA LOC100132354过表达载体的构建及鉴定  

Construction and identification of lentivirus-mediated long chain non-coding RNA LOC100132354 over expression vector

作　　者：胡丽娟[1] 陈洁[2] 章帆[1] 王君君[1] 陈坚[1] 王瑜敏[1] HU Li-juan CHEN Jie ZHANG Fan WANG Jun-jun CHEN Jian WANG Yu-min(Center for Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, Chin)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江温州325000 [2]温州医科大学附属第一医院ICU室,浙江温州325000

出　　处：《中国卫生检验杂志》2017年第18期2588-2592,共5页Chinese Journal of Health Laboratory Technology

基　　金：国家自然青年基金(81401736);浙江省自然基金(LQ1-6H160020);温州市科技局课题(Y20150097)

摘　　要：目的探讨慢病毒介导LOC100132354过表达载体的构建及鉴定。方法化学合成LOC100132354全基因序列,与GV303载体进行连接反应获得重组质粒;进一步制备感受态细胞,并行转化实验,PCR鉴定进行转化子,对阳性转化子进行测序;构建好的LOC100132354过表达载体转染至293T细胞包装病毒,采用荧光显微镜观察荧光情况,用q PCR检测LOC100132354的表达情况。结果酶切结果与预期一致,阳性转化子克隆测序结果与LOC100132354的RNA序列完全一致,293T细胞转染后荧光表达结果较强,q PCR结果显示293T细胞转染过表达载体后LOC100132354表达水平为对照组的555 427.148倍,差异有统计学意义(P<0.001)。结论成功构建了慢病毒介导LOC100132354过表达载体,为后续进一步研究提供了基础。Objective To investigate the construction and identification of lentivirus-mediated LOC100132354 over expression vector. Methods The full-length gene of LOC100132354 was synthesized and the recombinant plasmid was ligated with GV303 vector. Competent cells were compared and we then performed a transformation experiment,then transformants was identified by PCR and sequencing. The constructed LOC100132354 over expression vector was transfected into 293 T cells. The expression of LOC100132354 was detected by q PCR and fluorescence microscope. Results The restriction result of enzyme was consistent with expectation and sequencing of the positive clones was consistent with the RNA sequence of LOC100132354.Strong fluorescence expression was obtained after 293 T cell transfection. The result of q PCR showed that the expression level of LOC100132354 after 293 T cells transfection with the expression vector was 555 427. 148 times of that in the control group,with the differences statistically significant（ P〈0. 001）. Conclusion Lentivirus-mediated LOC100132354 over expression vector was constructed successfully,which would provide experimental basis for further study.

关 键 词：慢病毒 过表达载体 长链非编码RNA LOC100132354 

分 类 号：R734.2[医药卫生—肿瘤]