人ODC基因RNAi慢病毒载体的构建与鉴定

Construction and identification of the lentiviral RNA interference vector of human ornithine decarboxylase gene

目的:构建人鸟氨酸脱羧酶(ODC)基因的RNAi慢病毒载体。方法:根据ODC mRNA序列, 选择3个19nts的靶序列,设计并合成包含各正反义靶序列的互补DNA链,退火后插入pSuper载体的H1RNA启动子后产生pRiODC,将其中的ODC shRNA表达结构酶切插入慢病毒转移质粒pHR' CMVGFP,产生pHRiODC/GFP。与△R8.2和pCMVVSVG共转染HEK293T细胞,包装产生慢病毒,分别采用转导HEK293T细胞和Real-Time RT-PCR的方法测定慢病毒的功能滴度和病毒RNA分子拷贝数。结果:酶切和测序证实,pRiODC质粒构建正确,产生能同时表达GFP和转录产生ODC shRNA的慢病毒转移质粒PHRiODC/GFP,未浓缩及浓缩病毒悬液的功能滴度分别为6.3×104 TU/ml和7×106TU/ml, RNA拷贝数分别为3.4×108copies/ml和3.6×1010copies/ml。结论:成功构建人ODC基因RNAi慢病毒载体,为通过阻断ODC基因表达治疗恶性肿瘤奠定了基础。

Objective: To construct the lentiviral RNA interference (RNAi) vector of human ornithine decarboxylase (ODC) gene. Methods: Three target sequences were selected according to ODC mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After phosphorylation and annealing, these double strands DNA were cloned to Bgl Ⅱand Hind Ⅲ sites of pSuper. Then the product pRiODC was confirmed by electrophoresis and sequencing. Next, ODC shRNA was cloned to pHR' CMVGFP, a transfer vector of lentivirus, pHRi-ODC/GFP was produced. And it was cotransfected along with AR8.2 and pCMVVSVG into HEK293T to package lentivirus particles. The functional titer was determined by FCM after transduction in HEK293T cells. At the mean time, the viral RNA copies were quantified by Real-Time RT-PCR method. Results: It was confirmed by digestion and sequencing that ODC shRNA expression structure was correctly cloned to pSuper and pHR' CMVGFP respectively. The functional tiler of unconcentraled virus and concentrated virus were 6.3×104TU/ml and 7×106TU/ml respectively, and their RNA copies were 3.4×108 copies/ml and 3.6×1010 copies/ml. Conclusion: RNAi lenlivirus veclor of human ODC gene has been constructed, which is the essential building block required for the ODC related gene therapy research.