新型RNAi载体抑制HPV16E6基因在人宫颈癌细胞Caski中的表达

摘要

目的:利用自行构建的一种基于Semliki森林病毒的新型RNAi载体pSFV-RNAi Ready,验证将其用于短期高效沉默HPV16E6基因的效果.方法:以HPV16E6为靶标基因,设计并构建基于pSFV-RNAi Ready的重组质粒,分直接电击转染和病毒颗粒共培养两种方式转入人宫颈癌细胞株Caski,RT-PCR、Western blot检测HPV16E6表达水平.结果:重组质粒对HPV16E6沉默效果优于常规RNAi质料载体,接近化学合成小RNA,抑制率可高达90%以上,10天后效果仍然存在;结论:新型RNAi载体pSFV-RNAi Ready可较好地应用于特异高丰度靶基因的表达抑制,有望用于未来的科学研究或治疗应用.%Objective: To examine the silence effects of HPV 16E6 gene, which mediated by pSFV-RNAi Ready, a novel RNAi vector based on SFV replicator. Methods: Targeting HPV16E6 gene, Construct an re-combined plamid with pSFV-RNAi Ready, Then introduced into Caski cells by PLUS transfection and co-culture with viral particle. The expression of HPV16 E6 were investigated by RT-PCR and Western blot. Results: The novel pSFV-RNAi Ready vector was constructed successfully, Results of RT-PCR and Western blot indicated that pSFV-RNAi Ready mediated RNAi reduced HPV16E6 expression by more than 90 percent, which similar to chemically synthesized short interfering RNAs, much stronger than traditional RNAi vector, and continue to work att er more than 10 days.Conclusion: pSFV-RNAi Ready vector could silence the specific high-expressed target gene efficiently, It have potential for scientific research or clinlical therapy in the futrue.