TFEB基因shRNA慢病毒载体的构建及鉴定  

Construction and Identification of TFEB Gene shRNA Lentiviral Vector

作　　者：常熙昊 郭雅坤 李薇[1] CHANG Xihao;GUO Yakun;LI Wei(Inner Mongolia Medical University,Hohhot 010110,China)

机构地区：[1]内蒙古医科大学,内蒙古呼和浩特010110

出　　处：《生物化工》2023年第2期69-72,共4页Biological Chemical Engineering

基　　金：内蒙古医科大学科技百万工程项目(YKD2018KJBW015);内蒙古医科大学2022年大学生科技创新“英才培育”项目(YCPY2022154)。

摘　　要：目的:构建TFEB基因的shRNA慢病毒载体,并鉴定是否成功。采用该载体包装的慢病毒侵染所获得的细胞系可利用TFEB(转录因子EB)作为靶点,用于TFEB相关性疾病发病机制的研究及为临床疾病研究提供新的药物治疗方向。方法:利用PCR、双酶切、连接、转化等分子生物学技术构建TFEB基因的shRNA慢病毒载体,利用测序鉴定序列是否连接成功;通过RNAi技术,将构建成功的重组质粒包装成慢病毒并侵染细胞,建立敲低TFEB的细胞系,利用Western blot检测侵染及对照HeLa细胞TFEB蛋白表达水平。结果:测序结果显示pLKO.1-TFEB-shRNA重组质粒分别包含3U、OR不同干扰序列,重组质粒构建成功;Western blot检测结果表明,序列为3U和OR的慢病毒收集液侵染的细胞内TFEB表达量均降低。结论:利用序列为3U和OR的shRNA构建的重组质粒、包装的慢病毒均成功,干扰了侵染获得的细胞系中TFEB的表达。Objective:To construct shRNA lentiviral vector of TFEB gene and verify the success.The cell lines infected with lentivirus packaged with this vector can be used as the target of TFEB(Transcription Factor EB)to study the pathogenesis of TFEB-related diseases and provide a new direction of drug therapy for clinical disease research.Methods:The shRNA lentiviral vectors of TFEB gene are constructed by molecular biological techniques,such as PCR,double enzyme digestion,ligation and transformation,and is identified by sequencing method.The constructed recombinant plasmids are packaged into lentiviruses and infected with cells by RNAi technology,and cell lines of TFEB knockdown are established.The expression level of TFEB protein in infected and control HeLa cells are detected by Western blot.Results:The sequencing results show that the recombinant plasmids pLKO.1-TFEB-shRNA contained 3U,OR different interference sequences,respectively,and the recombinant plasmids are successfully constructed.The results of Western blot detection show that the expression of TFEB in the cells infected with lentivirus collection fluid with 3U and OR sequence are decreased.Conclusions:The recombinant plasmids constructed with shRNA 3U and OR,and the lentivirus packaged successful,interfering the expression of TFEB in the infected cell lines.

关 键 词：TFEB基因 shRNA慢病毒载体 RNAI技术 

分 类 号：Q782[生物学—分子生物学]