CAJ | 학술논문

目的：构建人类基因组中16个组蛋白乙酰化酶的短发夹 RNA（shRNA）慢病毒载体文库，为进一步探索表观遗传学基因在 HBV 调控中的作用机制提供有利工具。方法根据 shRNA 引物设计原则，针对每个基因选择8对确保干扰效率的 shRNA 序列（A～H），将引物退火后连接至 shRNA空慢病毒载体上转化，PCR 法验证菌落克隆，确认阳性克隆；对质量合格的质粒再进行单切酶验证。分别将同一基因的4个 shRNA 慢病毒质粒混合，分别包装病毒。293T 细胞转染48 h 和72 h 后收集病毒粗液，感染 HepG2．2．15细胞。感染72 h 后用荧光显微镜观察并评估荧光细胞比例。结果针对16个乙酰化酶基因，构建128个慢病毒载体 RNA 干扰（RNAi）文库，72 h 后慢病毒感染 HepG2．2．15细胞的效率＞80％。结论成功构建16个组蛋白乙酰化酶的 shRNA 慢病毒载体，从而为研究人组蛋白乙酰化酶对 HBV 复制的影响奠定了坚实的基础。
목적：구건인류기인조중16개조단백을선화매적단발협 RNA（shRNA）만병독재체문고，위진일보탐색표관유전학기인재 HBV 조공중적작용궤제제공유리공구。방법근거 shRNA 인물설계원칙，침대매개기인선택8대학보간우효솔적 shRNA 서렬（A～H），장인물퇴화후련접지 shRNA공만병독재체상전화，PCR 법험증균락극륭，학인양성극륭；대질량합격적질립재진행단절매험증。분별장동일기인적4개 shRNA 만병독질립혼합，분별포장병독。293T 세포전염48 h 화72 h 후수집병독조액，감염 HepG2．2．15세포。감염72 h 후용형광현미경관찰병평고형광세포비례。결과침대16개을선화매기인，구건128개만병독재체 RNA 간우（RNAi）문고，72 h 후만병독감염 HepG2．2．15세포적효솔＞80％。결론성공구건16개조단백을선화매적 shRNA 만병독재체，종이위연구인조단백을선화매대 HBV 복제적영향전정료견실적기출。
Objective To construct lentivirus vectors carrying 16 short hairpin RNA (shRNA) expression cassettes targeting histone acetyltransferases and provide a powerful research approach to explore the mechanism of epigenetic genes in regulating hepatitis B virus (HBV).Methods Following the rule of short shRNA primer design,eight-pair primers (A ~ H )for each gene,which had stable interfering efficiency,were designed.The annealed primers were connected to the empty lentiviral vectors of shRNA for transformation.In order to confirm the positive clones,clones were analyzed by real-time polymerase chain reaction (RT-PCR ).Then, qualified plasmids were verified by enzyme digestion technology.Four shRNA lentivirus plasmids against the same gene were mixed to build lentivirus respectively.After the virus transfected into 293T cells for 48 and 72 hours,supernatants were collected to infect HepG2.2.15 cells.The percentage of fluorescent cells were observed and assessed by microscope 72 hours after infection.Results One hundred and twenty-eight lentiviral vectors of RNA interference (RNAi)library were constructed against 16 histone acetyltransferases and more than 80% of HepG2.2.15 cells were infected with lentivirus 72 hours after infection.Conclusions Sixteen shRNA lentivirus vectors against histone acetyltransferase are successfully constructed.Thus,a solid foundation for the study of the effect of human histone deacetylase on HBV replication is established.