HDAC8过表达慢病毒载体构建、转染及表达的研究  

Construction, transfection and expression of HDAC8-gene-over-expressed lentivirus vector

作　　者：傅瑜[1] 张平[1] 张永栋[1] 单腾飞[1] 董伟杰[1] 江宏兵[1] 

机构地区：[1]南京医科大学口腔医学研究所.附属口腔医院口腔颌面外科,江苏南京210029

出　　处：《口腔生物医学》2012年第4期181-184,共4页Oral Biomedicine

基　　金：国家自然科学基金(81070810);江苏高校优势学科建设工程资助项目

摘　　要：目的:构建大鼠组蛋白去乙酰基转移酶(histone deacetylase 8,HDAC8)基因过表达的慢病毒载体,转染大鼠骨髓基质细胞(bone marrow stromal cells,BMSCs)后观察HDAC8的表达。方法:采用DNA重组技术将HDAC8基因插入到慢病毒表达载体质粒pGC-FU中,重组获得慢病毒载体pGC-FU-HDAC8。重组慢病毒载体经过测序鉴定后,转染293T细胞生产病毒液,用得到的病毒液转染大鼠BMSCs,实时荧光定量PCR和蛋白质免疫印迹法分析转染前后HDAC8表达情况。结果:测序结果证实HDAC8基因正确插入载体中,成功构建大鼠HDAC8基因过表达载体。大鼠BMSCs转染后HDAC8 mRNA及蛋白表达显著上调。结论:针对大鼠HDAC8基因过表达慢病毒载体构建成功,并能有效增强BMSCs中HDAC8基因的表达。Objective：To construct rat HDAC8-gene-over-expressed lentivirus vector and study expression of HDAC8 in bone marrow stromal cells （ BMSCs ） after transfection. Methods：The HDAC8 gene was inserted into plasmid pGC-FU of lentiviral vector by recombinant DNA technology. Lentivirus vector pGC-FU-HDAC8 was got after recombination. The The recombinant lentivirus vector was detected by DNA sequencing. The recombined plasmid pGC-FU-HDAC8 was transfected into 293 cell line. Virus yielded by 293T cell was transfected into rat BMSCs. The expression of HDAC8 was detected by Western bolt and real-time RT-PCR before and after transfection. Results：It was confirmed by DNA sequencing that the HDAC8 was correctly inserted into the vector, and that rat HDAC8-gene-over-expressed lentivirus vectors were successfully constructed. After transfection, the expression of HDAC8 was significantly upregulated either in mRNA level or in protein lever in rat BMSCs. Conclusions：The construction of HDACS-gene-over-expressed lentivirus vector was successful, and it efficiently up-regulated the expression of HDAC8 in rat BMSCs.

关 键 词：组蛋白去乙酰化酶8 过表达 表观遗传修饰 骨髓基质细胞 

分 类 号：Q786[生物学—分子生物学]