小鼠ameloblastin基因重组慢性病毒表达载体的构建及表达  

Construction and Expression of Recombinant Lentivirus Vector Expressing Mouse Ameloblastin Gene

作　　者：刘晓影[1] 林维平[1] 高文风[2] 赵小娟[1] 高志芹[1] 

机构地区：[1]潍坊医学院细胞生物学教研室,山东潍坊261042 [2]潍坊医学院临床医学系内科教研室,山东潍坊261042

出　　处：《现代生物医学进展》2008年第10期1829-1831,共3页Progress in Modern Biomedicine

摘　　要：目的:构建表达小鼠ameloblastin基因的慢性病毒表达载体,生产有感染及表达能力的病毒,为进一步研究ameloblastin基因在牙釉质形成中的功能奠定基础。方法:利用RT-PCR的方法从出生后1d钟状期小鼠牙胚组织totalRNA中扩增ameloblastin编码区cDNA,并克隆到pCRII-TOPO载体中,再亚克隆到pNL-IRES2-EGFP中。将病毒三质粒系统转染293T细胞,收集病毒,并鉴定病毒感染及表达能力。结果:通过酶切和PCR的方法鉴定ameloblastin基因已成功的构建入慢性病毒表达载体pNL-IRES2-EGFP中,经293T细胞生产的病毒感染人牙髓干细胞(DPSCs),DPSCs有较强荧光发出,并可稳定表达ameloblastin基因。结论:成功的构建了慢性病毒表达载体,并获得有感染及表达能力的病毒。Objective： To construct lentivirus vector expressing mouse ameloblastin gene,it will provide a foundation for studying the function ofameloblastin in further study. Methods： RT-PCR was performed to get mouse ameloblastin coding region cDNA from spe- cial total RNA which was obtained from tooth germ at the bell stage of postnatal lday, and coding region cDNA was cloned into pCRI- I-TOPO, then lentivirus vector pNL- IRES2- EGFP containing ameloblastin was constructed. The positive recombinant pNL-amelo- blastin-IRES2-EGFP were transfected into 293T cells, and collected lentivirus, moreover, identified their ability of infection. Results： pNL-ameloblastin-IRES2-EGFP was Constructed successfully, which was detected by restriction enzyme digestion analysis and PCR. Lentivirus produced in 293T infected human dental pulp stem cells （DPSCs）, then fluorescence protein and ameloblastin can be expressed stably in DPSCs. Conclusions： We have constructed recombinant lentivirus vector expressing mouse ameloblastin gene （pNL-ameloblastin-IRES2-EGFP ）,and produced lentivirus with ability of infection.

关 键 词：AMELOBLASTIN 载体构建 慢性病毒表达载体pNL—IRES2-EGFP 转染 

分 类 号：Q784[生物学—分子生物学] Q786