穿梭质粒pSP189/哺乳动物Vero细胞检测系统在喹乙醇诱变分子机制研究中的应用

Molecular Mechanism of Mutag-enesis Induced by Olaquindox Using a Shuttle Vector pSP189/Mammalian Cell System

背景与目的将短暂复制型穿梭质粒pSP189与非洲绿猴肾细胞(Vero细胞系)组成穿梭质粒/哺乳动物细胞诱变检测系统,并将该系统应用于喹乙醇的诱变性检测。材料与方法质粒经6.6μg.ml-1喹乙醇处理后,转染Vero细胞,从细胞中回收质粒转化E.coliMBM7070指示菌筛选突变体。结果喹乙醇处理组的诱变频率(24×10-4)明显高于溶剂对照组(1.2×10-4);试验进一步对61个突变子中的24个突变子质粒靶基因进行序列分析,发现喹乙醇引起质粒靶基因核苷酸序列改变主要表现为点突变和连续缺失,且以引发点突变为主,占总数的70%。点突变主要集中在GC碱基对,且包含在5'-NNTTNN-3'的序列中,其中的N位易发生突变,主要表现为GC-TA或GC-AT置换;而缺失以连续缺失为主,包含在含ANGGCCNAAA的序列中。结论喹乙醇能诱发质粒pSP189靶基因突变。

BACKGROUND ＆ AIM： Using a new SV40-based shuttle vector pSP189 and African green monkey kidney cells（Vero E6 cell line）, we constituted a shuttle vector/mammalian cell system to detect mutagenesis in vitro induced by olaquindox. MATERIAL AND METHODS： The vector plasmids were trealed by olaquindox , then transfected into Vero ceils. After recovery from ceils, the plasmids were transformed into MBM7070 and mutant colonies were selected. The mutation in 24 mutants was determined by dideoxy sequence analysis. RESULTS： The lesion induced by 6.6μg·ml^-1 olaqiunxox included a large number of base substitutions, in addition to tandem base deletion. Point mutation frequencies of modified plasmids in vitro were dramatically increased to 70% the spontaneous background level,and base substitutions concentrated on the G：C base pair, the predominant mutation was G：C-T：A or G：C-A：T. Olaqiundox-induced mutations on the SupF shuttle vector was not distributed randomly, they had mutation hot spots （155bp） and sequence specificity, which were contained in the sequence 5＇ -NNTTNN-3＇ Mutation was common in N site, tandem base deletion or rearrangement focused on ANGGCCNAAA sequence. CONCLUSION： Olaquindox could induce plasmid shuttle vector mutagenesis.