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

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Age Ⅰ和EcoR Ⅰ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2×10s 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.

Construction and the infection efficiency of BimS lentivirus RNA interference vector

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 annealed into double-stranded oligo sequences,and then connected with vector linearized with Age Ⅰ and EcoR Ⅰ enzyme.The recombinant 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 × 108 TU/ml,MOI =20,and the transfection efficiency was 85％.The BmS mRNA relative expression of pFU-GV-BmS-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 interference vectors can be constructed,and can efficiently infect ACC-2 cells.