摘要
目的:针对小鼠烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)基因构建质粒并进行慢病毒包装,同时检测其表达水平和干扰效率,为进一步探讨该基因的功能提供研究工具和实验基础。方法:根据NMNAT1基因信息,用慢病毒载体pLenti6构建三种重组质粒,分别包含NMNAT1 cDNA全长、一个针对NMNAT1的小干扰序列和一个用于干扰对照的阴性序列。把这些质粒包装进慢病毒载体,并检测病毒滴度,再用慢病毒感染Hela细胞检测NMNAT1表达量和RNA干扰效率。结果:测序结果证明目的序列正确地插入到载体内。通过qPCR方法鉴定慢病毒包装成功,病毒滴度均为2×108 TU/ml以上。表达NMNAT1的重组慢病毒感染Hela细胞,该细胞能够高水平表达NMNAT1蛋白,而携带RNAi序列的慢病毒能够显著抑制其表达,干扰效率在70%以上。结论:针对NMNAT1的过表达和RNAi重组慢病毒制备成功,为进一步研究NMNAT1基因的功能和用慢病毒进行基因治疗提供了良好的研究工具。
Objective: To construct two recombinant lentiviral vetcors carring mouse NMNAT1 gene and RNAi targeting NMNAT1. Methods: According to GenBank,the full-length cDNA sequence of mouse NMNAT1,an interfering sequence targeting NANAT1 and a negative sequence were designed,synthesized and inserted into plasmid pLenti6 lentiviral vector.The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells.The virus titer was tested by qPCR methods.After infection of Hela cells with these lentiviruses,the expression of NMNAT1 was detected by qPCR and Western blot. Results: All the recombinant plasmids were confirmed by sequencing.The titer of virus was over 2×108 TU/mL.Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1.The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence. Conclusions: The lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NANAT1 have been successfully constructed.
出处
《浙江大学学报（医学版）》
CAS
CSCD
北大核心
2011年第6期622-629,共8页
Journal of Zhejiang University(Medical Sciences)
基金
黑龙江省自然科学基金资助项目(D200980)
