BimS慢病毒RNA干扰载体的构建及其感染效率和干扰效果的实验研究

发布时间：2018-05-27 14:11

本文选题：RNA干扰 + BimS基因　；参考：《实用口腔医学杂志》2017年01期

【摘要】：目的:应用RNA干扰技术构建BimS慢病毒RNA干扰载体,探讨其感染效率和干扰效果。方法:针对人BimS设计3个干扰靶点,将单链引物退火成双链oligo序列,连接入AgeI和EcoRI双酶切线性化载体中。将重组质粒进行病毒包装、并且通过感染ACC-2细胞观察其感染效率,定量PCR检测其对BimS mRNA干扰效果。结果:PCR产物经扩增电泳后阳性克隆得到337bp条带,插入序列片段与DNA测序结果完全相符。重组慢病毒载体在293T细胞中包装获得滴度为2×10~8 TU/ml的病毒颗粒,MOI=20,转染效率为85%。转染pFU-GV-BmS-1组、pFU-GV-BmS-2组及对照组BmS mRNA相对表达水平分别为:0.743±0.025、0.466±0.023、1.266±0.042(组间两两比较,P0.05)。结论:BimS慢病毒RNA干扰载体构建成功,并能高效感染ACC-2细胞及下调BmS mRNA表达。
[Abstract]:Objective: to construct BimS lentivirus RNA interference carrier by using RNA interference technique, and to explore its infection efficiency and interference effect. Methods: 3 interference targets were designed for human BimS. Single chain primers were annealed into double chain oligo sequences and joined into AgeI and EcoRI double enzyme tangential linearized carriers. The recombinant plasmid was packaged by the recombinant plasmid and infected by ACC-2 cells. The infection efficiency was observed and the interference effect on BimS mRNA was detected by quantitative PCR. Results: the 337bp bands were obtained by the positive clones of the PCR products after amplification electrophoresis. The inserted sequence fragments were completely consistent with the results of DNA sequencing. The recombinant lentivirus vector was packed in 293T cells to obtain 2 x 10~8 TU/ml virus particles, MOI=20, the transfection efficiency was 85%. transfection pF. The relative expression levels of BmS mRNA in group U-GV-BmS-1, group pFU-GV-BmS-2 and control group were 0.743 + 0.025,0.466 + 0.023,1.266 + 0.042 (22 comparison between groups, P0.05). Conclusion: the RNA interference vector of BimS lentivirus was constructed successfully, and it could efficiently infect ACC-2 cells and downregulate BmS mRNA table.
【作者单位】：西安交通大学口腔医院口腔颌面外科;
【基金】：陕西省社发攻关资助项目(编号:2013k12-03-21)
【分类号】：R739.8

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