小鼠GATA-3基因RNA干扰慢病毒载体的构建与鉴别

发布时间：2018-04-16 03:22

本文选题：鼻炎 + 变应性　；参考：《临床耳鼻咽喉头颈外科杂志》2017年08期

【摘要】：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。
[Abstract]:Objective: to explore the construction and identification of lentivirus vector which interferes with mouse GATA-3 gene.Methods: the coding region of GATA-3 gene in mouse mononuclear cells was analyzed and three target interference sequences with optimal kinetic parameters were designed and synthesized.The synthesized single-stranded oligo was spliced by PCR technique. The synthesized sequence was inserted into the shRNA lentivirus vector and transformed into the competent cell DH5 伪. After sequencing and identifying the gene sequence in the recombinant clone, the shRNA-GATA-3 expression vector was constructed.According to different interference sequences, lentivirus vectors were divided into 4 groups: 1: siRNA-685 (LV3-GATA-3-Mus-685)) group (LV3-GATA-3-Mus-1152)) and control group.The shRNA vector and the expression vector were cotransfected into 293T cells, and the optimal interference sequence was screened by qPCR detection.The best LV3-GATA-3-Mus-1615 interference sequence was constructed into lentivirus vector. The vector was co-transfected into 293T cells. The virus was packaged, the virus solution was collected, the concentration was determined by ultracentrifugation, and the titer was measured.Results: cloning and sequencing proved that lentivirus vector constructed successfully. SiRNA-1615 group GATA-3mRNA relative expression was 0.004) compared with siRNA-685 group 0.009 siRNA-1152 group (0.009) and control group (0.022) significantly reduced LV3-GA-3-Mus-1615 interference sequence.The titer of the collected virus supernatant was 5 脳 10 ~ (8) TU / ml.After 48 hours of transfection, the transfection was observed successfully by fluorescence microscope.Conclusion: the murine GATA-3 gene RNAi lentivirus vector was successfully constructed and identified.
【作者单位】：昆明医科大学第一附属医院耳鼻咽喉头颈外科;中国科学院昆明动物研究所动物模型与人类疾病机理重点实验室;
【基金】：云南省自然基金资助项目(No:2011FZ120)
【分类号】：R765.21

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