慢病毒介导的犬WRD细胞p53基因沉默及稳定感染细胞系的建立

发布时间：2018-03-19 17:35

本文选题：p　切入点：RNA干扰　出处：《南方医科大学学报》2014年12期 　论文类型：期刊论文

【摘要】：目的构建靶向犬p53基因慢病毒干扰载体,建立稳定沉默p53基因的WRD/p53-细胞系。方法设计并构建4条针对犬p53基因的特异性shRNA干扰质粒。将构建的慢病毒表达载体(p GMLV-p53)和包装质粒(packaging mix)组成的包装系统共转染293T细胞,包装病毒,收集病毒原液,超滤浓缩,并测定滴度。包装好的慢病毒感染WRD细胞,Western blot和实时荧光定量PCR方法检测不同靶点RNA干扰载体对p53基因的干扰效果,确定有效靶点。针对有效靶点慢病毒颗粒以最适感染复数(multiplicity of infection,MOI)感染WRD细胞,puromycin抗生素筛选稳定感染细胞系WRD/p53-。结果结果显示p53 RNAi慢病毒载体构建成功,成功包装四种不同靶点的p53基因RNA干扰慢病毒,病毒滴度均达到1×109TU/ml。四种干扰载体慢病毒均具有较好的干扰效果,选用沉默效果最好的p GMLV-p53A1为最优靶点,成功建立p53 RNAi慢病毒载体稳定感染细胞系WRD/p53-。结论成功构建p53 RNAi慢病毒载体,并有效干扰WRD细胞中p53 mRNA和蛋白表达,同时成功筛选并建立p53RNAi慢病毒稳定感染WRD/p53-细胞系。
[Abstract]:Objective to construct the targeting vectors of canine p53 gene lentiviral interference, to establish stable WRD/p53- cell line gene silencing by p53. Methods the design and construction of 4 against canine p53 gene specific shRNA interference plasmid. The constructed lentiviral expression vector (P GMLV-p53) (packaging Mix) and packaging plasmids were transfected into 293T packaging systems consisting of packaging cell, virus, virus stock solution was collected and concentrated by ultrafiltration, and the titer of lentivirus infection. Packaged in WRD cells, Western blot detection and real-time fluorescence quantitative PCR method of jamming effect on p53 gene targeting different RNA interference vector, determine the effective target. The effective target lentiviral particles to the optimal multiplicity of infection (multiplicity of infection, MOI) infection of WRD cells, puromycin cells infected with WRD/p53-. stable antibiotic screening results showed that p53 RNAi lentivirus vector was constructed successfully, four different target packaging success The p53 gene RNA interference lentiviral virus titer reached 1 * four 109TU/ml. interference lentivirus vector has better interference effect, the silencing effect of the best p GMLV-p53A1 is the best target, successfully constructed p53 RNAi lentivirus vector stably infected WRD/p53-. cell line objective to construct p53 RNAi lentivirus vector, and effective jamming the expression of p53 mRNA and protein in WRD cells, and successfully screened and established p53RNAi lentivirus infected WRD/p53- stable cell lines.

【作者单位】：宁夏医科大学总院呼吸与危重医学科;宁夏医科大学生物化学与分子生物学系;宁夏医科大学生育力保持教育部重点实验室;
【基金】：国家自然科学基金(81060130)~~
【分类号】：R373

【共引文献】