靶向人乳头瘤病毒18E6的短发夹结构RNA慢病毒载体的构建

Construction of a shRNA Lentiviral Vector Aiming at the Target of HPV18E6 Oncogene

目的:构建含有靶向人乳头瘤病毒18型E6癌基因(HPV18E6)的短发夹结构RNA(shRNA)的慢病毒载体。方法:根据HPV18E6癌基因的相关信息,设计并合成shRNA干扰序列,连入pGCSIL-GFP质粒,构建并鉴定含有shRNA的重组质粒pGC-shRNA;将重组质粒pGC-shRNA和病毒包装质粒pHelper1.0、pHelper2.0共转染293T细胞,进行病毒包装,收集病毒液并进行浓缩、纯化;所得病毒液感染293T细胞,测定病毒滴度,并验证其对宫颈癌HeLa细胞HPV18E6癌基因的干扰效果。结果:HPV18E6的shRNA干扰序列与人类基因组没有同源性,此插入序列转录后得到的RNA二级结构能形成发夹状结构;重组质粒pGC-shRNA经PCR鉴定及DNA测序结果显示该重组质粒构建成功;成功对病毒进行包装、浓缩及纯化后,所得病毒滴度为3×108TU/mL,并能很好地抑制HPV18E6癌基因的表达。结论:成功构建了HPV18E6-RNAi-LV慢病毒载体,为后续RNA干扰研究的开展提供了工具。

Objective：To construct a lentiviral vector that contains the short hairpin RNA（shRNA） interfering sequence aiming at the target of human papillomavirus 18E6（HPV18E6） oncogene.Methods：Based on the information of HPV18E6 oncogene,shRNA interfering sequence was designed and synthesized,and was inserted into pGCSIL-GFP plasmid to construct a recombination plasmid pGC-shRNA which contains shRNA interfering sequence.Recombination plasmid and packaging plasmid pHelper 1.0 and pHelper 2.0 co-transduced the 293T cells to package the lentiviral vector.The lentiviral vector solution was collected,concentrated and purified.The lentiviral vector solution transduced 293T cells to determine the concentration of the lentiviral vector solution after concentration and purification.At last,the interfering effect for HPV18E6 oncogene with the lentiviral vector transducing HeLa cells was tested.Results：Indexing shRNA of interfering sequence aiming at the target of HPV18E6 oncogene with human genes in BLASTER,there was no homogeneous,and the second constructure of the transcripts of interfering sequence can be conformed short hairpin.The recombination plasmid pGC-shRNA was obtained with the correct sequence in the insertion after the appraisal.After packaging,collecting and concentrating the lentiviral vector successfully,the concentration of lentiviral vector solution was 3×108 TU/mL and the recombination lentiviral vector can restrain the expression of HPV18E6 oncogene remarkably.Conclusion：The lentiviral vectors of HPV18E6-RNAi-LV were constructed successfully,which provided practical tool for later study.