狨猴B2m基因沉默位点在细胞水平的验证

发布时间：2018-04-16 13:07

  本文选题：基因沉默 + 狨猴　； 参考：《中国比较医学杂志》2017年05期

【摘要】：目的在细胞水平筛选狨猴B2m基因的有效沉默靶点,并进行验证。方法查询人源B2m验证过的有效siRNA靶位点序列,与狨猴B2m基因序列进行同源性比较,选择匹配靶点合成shRNA序列。将体外合成的2条干扰序列分别与慢病毒载体FUGW-TDT连接,构建FUGW-TDT-shb2m干扰表达质粒,在聚乙烯亚胺(polyethylenimine,PEI)介导下转染293T细胞,转染后48h,用实时荧光定量法检测转染细胞中B2m基因mRNA的水平。结果筛选出2个与狨猴完全同源的B2m沉默靶位点,分别位于B2m mRNA的290~310 bp,665~685 bp;B2m两个靶点在转录水平的沉默效率分别为(46.54±7.91)%(P0.05)和(83.22±4.37)%(P0.0001),差异有显著性。结论成功构建成FUGW-TDT-shb2m重组质粒;在细胞水平筛选得到2个有效的B2m基因沉默靶点;为后续有关介导狨猴B2m基因沉默的研究奠定了基础。
[Abstract]:Objective to screen and verify the effective silencing target of marmoset B 2m gene at cell level.Methods the sequence of effective siRNA site verified by human B2m was searched, and the homology of B2m gene sequence of marmoset was compared with that of marmoset. The matching target was selected to synthesize shRNA sequence.The two interference sequences were ligated with lentivirus vector FUGW-TDT in vitro, and FUGW-TDT-shb2m interference expression plasmid was constructed and transfected into 293T cells mediated by polyethylene polyethylenimine (PEI). The mRNA level of B2m gene in transfected cells was detected by real-time fluorescence quantitative method at 48h after transfection.Results two B2m silencing sites homologous to marmosets were screened. The silencing efficiency of the two targets at the transcription level was 46.54 卤7.91 (P 0.05) and 83.22 卤4.3737 (P 0.0001), respectively. The silencing efficiency of the two targets was 46.54 卤7.91 and 83.22 卤4.37P0.0001, respectively.Conclusion the recombinant plasmid of FUGW-TDT-shb2m was successfully constructed, and two effective targets of B2m gene silencing were obtained at the cell level, which laid a foundation for the further study on the mediation of marmoset B2m gene silencing.
【作者单位】： 中国医学科学院医学实验动物研究所;北京大学生命科学学院生物膜及膜生物工程国家重点实验室北京大学麦戈文脑研究所;
【基金】：国家科技支撑计划(2014BAI03B01)
【分类号】：R-332

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