lncRNA4.9干扰慢病毒载体的构建

Construction of lncRNA4.9 interfering lentivirus vector

目的构建人巨细胞病毒(human cytomegalovirus,HCMV)转录的长链非编码RNA4.9(long non-coding RNA4.9,lncRNA4.9)干扰慢病毒载体。方法设计并合成3条以lncRNA4.9为靶点的干扰序列,构建穿梭质粒GV248和目的干扰序列的重组质粒,测序验证序列,将重组质粒与骨架质粒pHelper1.0、pHelper2.0共转染293T细胞,收集病毒颗粒并检测拷贝数。将干扰慢病毒载体转染THP-1细胞,观察荧光表达量,通过real-time RT-PCR法检测干扰效率。结果构建的3组慢病毒干扰载体(LV1、LV2、LV3),LV2和LV3具有干扰效率,LV2组干扰效率最高。结论成功构建lncRNA4.9干扰慢病毒载体,为后续进一步研究lncRNA4.9的功能奠定了基础。

Objective To construct an interfering lentivirus vector of long non-coding RNA 4.9(lncRNA4.9)transcribed by human cytomegalovirus(HCMV).Methods Three interfering sequences targeting lncRNA 4.9 were designed and synthesized.The shuttle plasmid GV248 and the target interfering sequence were combined and constructed.The recombinant plasmid was co-transfected with the skeleton plasmids pHelper1.0 and pHelper2.0 to 293T cells.Viral particles were collected and copies were determined.The interfering lentivirus vector was transfected into THP-1 cells to observe the fluorescence expression,and the interfering efficiency was detected by real-time RT-PCR.Results Three groups of lentivirus interference vectors(LV1,LV2,LV3)were constructed,LV2 and LV3 can interfere with the expression of lncRNA4.9,and the interference efficiency of LV2 group was the highest.Conclusions The interfering lentivirus vector of lncRNA 4.9 was successfully constructed,which laid a foundation for further study on the function of lncRNA 4.9.