TREM2基因过表达慢病毒载体的构建及其表达的研究

摘要

Objective:To construct lentiviral vector of a mouse triggering receptor expressed on myeloid cells-2,TREM2) gene over-expression,and examine the expression level of TREM2 in vitro.Methods:The TREM2 gene fragment was amplified by PCR,and the amplified product was ligated to the linearized GV358 vector.After identifying with PCR and DNA sequencing,the PCR product was co-transfected with the packaging plasmid into 293T cells and the virus titer was determined by drug screening method.293T cells were infected with recombinant lentivirus.GFP expression was observed and the expression level of TREM2 was detected by real-time quantitative PCR.Results:The TREM2 gene fragment was successfully obtained by PCR and ligated to the GV358 vector.TREM2-GV358 was confirmed to carry the correct TR(E)M2 gene by PCR and DNA sequencing.The virus was packaged in 293T cells and the virus titer was 2.0 ×108TU/mL.A large number of significant fluorescent cells infected by recombinant lentivirus were observed.Real-time quantitative PCR showed that the expression level of TREM2 was significantly increased(P＜0.05),when 293T cells was infected by lentivirus of TREM2 over-expression.Conclusion:The lentiviral vector over-expressing TREM2 gene was successfully constructed,which provides an experimental foundation for further studies on the functions of TREM2.%目的:构建小鼠编码2型髓样细胞触发受体(TREM2)基因过表达慢病毒载体,并且对其体外表达目的基因的水平进行检测.方法:采用PCR扩增TREM2基因片段,将扩增产物连接至经AgeI酶切后的线性化GV358载体;经PCR方法鉴定及DNA测序比对分析后,将其和包装质粒共转染至293T细胞并用药筛法行滴度测定.然后用重组慢病毒感染293T细胞,观察GFP表达并用实时定量PCR检测TREM2的表达水平.结果:通过PCR成功获得TREM2基因片段并连接到GV358病毒载体上,通过PCR及DNA测序鉴定均证实TREM2-GV358携带有正确的TREM2基因;在293T细胞中包装获得病毒并用药筛法测定病毒滴度为2.0×108 TU/mL.感染293T细胞后可观察到大量明显的荧光细胞;实时定量PCR检测结果显示,TREM2过表达慢病毒感染293T细胞后TREM2的表达水平明显增加(P＜0.05).结论:成功构建TREM2-GV358慢病毒载体,为TREM2基因的后期研究提供了实验基础.