组蛋白赖氨酸转移酶KAT6 B基因shRNA慢病毒载体的构建与鉴定

摘要

Objective The aims of this study were to construct short hairpin RNA(shRNA)lentiviral vector in breast cancer T47D cells,to carry out RNA interference on lysine acetyltransferase 6B(KAT6B/MORF)gene,to down-regulate its expression and to explore its function.Methods Two pairs of single-stranded short hairpin RNA(shRNA5 and shRNA8)and the corresponding control sequences(Scramble5 and Scramble8)were synthesized based on the CDS of KAT6B gene.Polymerase chain reaction(PCR) was used to amplify double-stranded and ligated with the entry vector(pENTR/pSM2(CMV)GFP),which were subjected to a doub-le digestion(EcoRl and Xhol)linearization and homologous recombination with the entry vector(pENTR/pSM2(CMV)GFP)to obtain an entry clone containing the desired fragment.The target fragment was recombined onto the target vector(pLenti x1 puro DEST)via the LR cloning reaction of the Gateway system.The lentiviral packaging plasmids were co-transfected into HEK-293T cells with two pairs of target plasmids. The supernatant of HEK -293T cells was collected and transformed into T47D cells. The expression of KAT6B protein was detected in T47D cells by Western blot.Results The single colony obtained from the transformation was identi-fied by sequencing,which was consistent with the target sequence,indicating that the lentiviral vector had been successfully construc-ted.The expression of KAT6B protein was significantly lower in the shRNA KAT6B group than that in the control group,which indica-ted that the constructed gene silencing vector could play a role in the KAT6B gene in T47D cells.Conclusion The shRNA lentiviral gene silencing vectors of KAT6B were constructed and identified in T47D cells,which indicated that the foundation for further study%目的 在乳腺癌细胞T47D中利用构建的短发卡RNA(shRNA)慢病毒载体对赖氨酸乙酰转移酶6B(KAT6B/MORF)基因进行RNA干扰,通过下调该基因的表达来研究其对乳腺癌细胞的抑制功能.方法 针对KAT6B基因的CDS区合成2对单链的短发卡RNA(shRNA5、shRNA8)及相应的对照组序列(Scramble5、Scramble8),通过聚合酶链式反应(PCR)扩增后,与双酶切(EcoRl、Xhol)线性化处理的入门载体(pENTR/pSM2(CMV)GFP)同源重组,获得含有目的片段的入门克隆.再通过Gateway系统的LR克隆反应,将目的片段重组到目的载体(pLenti x1 puro DEST)上.利用慢病毒包装系统,将慢病毒包装质粒分别与构建好的两对目的质粒共转染到HEK-293T细胞中,收集病毒上清,转导乳腺癌细胞T47D.最后,通过免疫印迹法(Western blot)检测KAT6B的表达.结果 对转化获得的单菌落进行测序鉴定,与目标序列完全一致,表明已经成功构建慢病毒载体.Western blot结果显示KAT6B基因的蛋白表达量在每组shRNA中都比相应的对照组显著降低,表明构建的基因沉默载体在乳腺癌细胞T47D中对KAT6B基因具有基因沉默作用.结论 本研究通过构建KAT6B基因的shR-NA慢病毒基因沉默载体,在乳腺癌细胞T47D中对KAT6B基因进行RNA干扰的研究,为进一步研究KAT6B基因在乳腺癌中发挥肿瘤抑制作用奠定了基础.