NMNAT1基因慢病毒载体的构建与鉴定

Construction and identification of lentiviral vector targeting NMNAT1 gene

目的:构建烟酰胺单核苷酸腺苷酰转移酶-1(NMNAT1)基因慢病毒载体,为其体内外实验研究提供依据.方法:将靶向NMNAT1基因连接到慢病毒载体pGC-E3/Lenti中,获得pGC-E3-NMNAT1统一质粒,经PCR检测以及测序鉴定后,与慢病毒包装质粒通过LipofectamineTM2000共转染至包装细胞293T,包装产生病毒液,并测定其滴度.结果:PCR扩增和测序结果证实,Nmnat1核苷酸链序列插入正确,包装慢病毒产生病毒悬液的滴度为1×105U/L.结论:成功构建NMNAT1基因慢病毒载体,为研究其在面神经损伤修复中的功能和基因治疗奠定了基础.

AIM： To construct a lentiviral vector targeting nicotinamide mononueleotide adenylyhransferase 1 （ NMNAT1 ） gene and provide the basis for further experiments in vivo and in vitro. METHODS：A NMNAT1 gene was cloned into lentiviral expression vector pGC-E3/Lenti. The recombinant vector pGC-E3/Len- ti-NMNAT1 confirmed by PCR and sequencing was cotransfected with the packaging plasmids into 293T cells by LipofectamineTM 2000. Virus in the supernatant was collected and the virus titer was measured. RESULTS： PCR and DNA sequencing demonstrated that the inserted sequences were correct. The titer of virus was 1 × 10^5 U/L. CONCLUSION： The lentivirus vector targeting NMNAT1 has been successfully constructed, which will provide a tool for the further study on the function of NMNAT1 gcne in the facial nerve injury and gene therapy.