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目的:探索干扰小鼠GATA-3基因的慢病毒载体的构建与鉴别方法。方法:分析小鼠单个核细胞内GATA-3基因的编码区,设计并合成3条最佳动力学参数靶点干扰序列的单链oligo。利用PCR技术对合成的单链oligo进行拼接反应,将合成好的序列装入shRNA慢病毒载体并转化至感受态细胞DH5α,测序并鉴定重组克隆中的基因序列后构建shRNA-GATA-3表达载体。根据不同的干扰序列将慢病毒载体分为4组:siRNA-685(LV3-GATA-3-Mus-685)组、siRNA-1152(LV3-GATA-3-Mus-1152)组、siRNA-1615(LV3-GATA-3-Mus-1615)组和对照组。将shRNA载体分别与表达载体共转染入293T细胞,通过qPCR检测筛选出最优干扰序列。将筛选到最佳的LV3-GATA-3-Mus-1615干扰序列构建入慢病毒载体,用构建的慢病毒干扰载体和包装质粒共转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,并测定滴度。结果:克隆测序验证无误,慢病毒载体构建成功。siRNA-1615组GATA-3mRNA相对表达量(0.004)与siRNA-685组(0.009)、siRNA-1152组(0.009)和对照组(0.022)相比明显减少,LV3-GATA-3-Mus-1615为最佳的干扰序列。收集的病毒上清测定病毒的滴度为5×10~8 TU/ml。转染实验48h后,荧光显微镜观察转染成功。结论:成功构建并鉴定小鼠GATA-3基因RNAi慢病毒载体。
Objective: To explore the construction and identification of lentiviral vector that interferes with GATA-3 gene in mice. Methods: The coding region of GATA-3 gene in mouse mononuclear cells was analyzed. Three single-stranded oligo of the best kinetic parameters were designed and synthesized. The synthesized single-stranded oligo was spliced by PCR. The synthesized sequence was inserted into the shRNA lentiviral vector and transformed into competent cells DH5α. The sequence of the recombinant clones was sequenced and identified, and the shRNA-GATA-3 expression vector was constructed . The lentiviral vectors were divided into four groups according to different interference sequences: siRNA-685 (LV3-GATA-3-Mus-685), siRNA-1152 LV3-GATA-3-Mus-1615) group and the control group. The shRNA vector was co-transfected into 293T cells with the expression vector, and the optimal interference sequence was screened by qPCR. The optimal LV3-GATA-3-Mus-1615 interference sequence was screened into lentiviral vector, 293T cells were co-transfected with the constructed lentiviral vector and packaging plasmid, the virus was packaged, the virus stock was collected, concentrated by ultracentrifugation, Titers were determined. Results: Clone sequencing verified that the lentiviral vector was successfully constructed. Compared with siRNA-685 group (0.009), siRNA-1152 group (0.009) and control group (0.022), the relative expression level of GATA-3 mRNA in siRNA-1615 group was significantly decreased The best interference sequence. The collected virus supernatant was assayed for virus titer of 5 × 10 -8 TU / ml. Forty-eight hours after transfection, the cells were transfected successfully by fluorescence microscopy. Conclusion: The RNAi lentiviral vector of mouse GATA-3 gene was successfully constructed and identified.