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,P<0.05)。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。
Objective： To construct BimS lentivirus RNA interference（RNAi） vector and to study its infection efficiency by using RNAi technique. Methods： Three interference targets were designed according to the BimS sequence. The single chain primer was an- nealed into double-stranded oligo sequences, and tilen connected with vector linearized with Age I and EcoR I enzyme. The recombi- nant plasmid was packaged, and the infection efficiency was observed by infecting ACC-2 cells. Results： After amplification, a 337 bp band was appeared in the electrophoresis results of positive clones. Sequence of inserted fragments were identical with the result of DNA sequencing. Restructuring lentivirus was packed in 293T cells, the virus titer was 2 ~ l0s TU/ml, MOI = 20, and the transtbction efficiency was 85%. The BmS mRNA relative expression of pFU-GV-BINS-1, pFU-GV-BMS-2 and control group was 0. 743 ± 0. 025, 0. 466 ± 0. 023 and 1. 266 ± 0. 042 respectively（ between each 2 groups, P 〈 0.05 ）. Conclusion ： BimS lentivirus virus RNA interfer- ence vectors can be constructed, and can efficiently infect ACC-2 cells.
作者
姚佳
饶国洲
李旭奎
YAO Jia RAO Guozhou LI Xukui(710004, Department of Oral and Maxillofacial Surgery, Stomalogical Hospital of Medical College , Xi＇an Jiaotong University, China)
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2017年第1期36-40,共5页
Journal of Practical Stomatology
基金
陕西省社发攻关资助项目(编号:2013k12-03-21)
