鞘氨醇激酶1干扰慢病毒载体及干扰慢病毒的构建

Construction of interference lentiviral vector and interference ientivirus targeted for sphingosine kinase 1

目的 构建人鞘氨醇激酶1（SPK1）干扰慢病毒载体,并制备SPK1干扰慢病毒.方法 将1 ～5号pLKO.2-shSPK1质粒及pLKO.1-Scramble-RFP对照质粒转染人胚肾293（HEK293）细胞,实时定量聚合酶链反应（qRT-PCR）检测pLKO.2-shSPK1质粒对SPK1的干扰效率,筛选干扰效果最好的pLKO.2-shSPK1质粒;将所筛选质粒中以SPK1为靶点的短发夹结构RNA（SPK1-shRNA）插入pLKO.1-Scramble-GFP质粒中,获得表达shRNA-SPK1的pLKO.1-shSPK1-GFP重组质粒;将该质粒进行测序及酶切鉴定后,转染HEK293细胞48 h后荧光显微镜及流式细胞仪观察绿色荧光强度,qRT-PCR检测其对SPK1的干扰效率.将pLKO.1-shSPK1-GFP重组质粒进行慢病毒包装及滴度检测.结果 与pLKO.1-Scramble-RFP对照质粒比较,转染1～5号pLKO.2-shSPK1质粒的HEK293细胞中SPK1 mRNA水平均有一定程度降低,其中3号pLKO.2-shSPK1质粒干扰效果最好,选取其进行pLKO.1-shSPK1-GFP重组质粒构建,经测序及酶切检验,证明重组质粒构建成功.pLKO.1-shSPK1-GFP重组质粒转染HEK293细胞后,绿色荧光阳性率达94.4％,SPK1 mRNA水平明显下降（P＜0.01）,干扰率达51.0％.进行慢病毒包装后测得病毒滴度为6×10^8 pfu/ml.结论 本研究成功构建了人SPK1干扰慢病毒载体,并制备了SPK1干扰慢病毒.

Objective To construct a interference lentiviral vector and a interference lentivirus targeted for sphingosine kinase 1 （SPK1）.Methods No.1-5 pLKO.2-shSPK1 and pLKO.1-Scramble-RFP plasmid were transfected into Human embryo kidney 293 cells （HEK293）,the interference efficiency for SPK1 was detected by real time-polymerase chain reaction （qRT-PCR）.The pLKO.2-shSPK1 with the best interference efficiency was selected for construction of pLKO.1-shSPK1-GFP vector by inserting short hairpin structure RNA targeted for SPK1 （shRNA-SPK1） into pLKO.1-Scramble-GFP plasmid.After identified by gene sequencing and enzyme digestion,the pLKO.1-shSPK1-GFP vector was transfected into HEK293 cells for 48 h,then the intensity of green fluorescent was observed by fluorescence microscope and flow cytometry,the interference efficiency for SPK1 was detected by qRT-PCR.At last,the lentivirus packaging was performed and the titer was detected.Results Compared with pLKO.1-Scramble-RFP,No.1-5 pLKO.2-shSPK1 all significantly reduced the SPK1 mRNA level in HEK293,among them,No.3 pLKO.2-shSPK1 obtained the best inference effect and was selected for construction of pLKO.1-shSPK1-GFP.The pLKO.1-shSPK1-GFP vector was demonstrated to be successfully constructed by gene sequencing and enzyme digestion.After transfecting pLKO.1-shSPK1-GFP into HEK293 cells,flow cytometry showed that the positive rate of green fluorescent protein in HEK293 reached 94.4%,and the mRNA level of SPK1 was significantly reduced （P 〈 0.01）,with interference efficiency of 51.0%.The lentiviral particle was packaged with a virus titer reaching 6 × 10^8 pfu/ml.Conclusion The study successfully constructed a interference lentiviral vector and a interference lentivirus targeted for SPK1.