穿梭质粒pSP189/哺乳动物Vero细胞诱变检测系统的研究及应用
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摘要
本研究将基于SV40病毒的短暂复制型穿梭质粒pSP189与非洲绿猴肾Vero细胞组成穿梭质粒／哺乳动物细胞诱变检测系统，并将该系统应用于兽药喹乙醇和喹烯酮的诱变性检测。本研究经阳离子脂质体转染法将pSP189质粒转染Vero细胞后，回收质粒转化宿主菌，获得每皿多于50个的转化子和1.9×10~(-4)的自发突变频率，试验结果表明该系统符合穿梭质粒诱变测试系统的要求。本研究用已知的直接阳性诱变物MNNG和间接阳性诱变物B(α)P分别对已建立的系统进行了验证，试验结果表明，MNNG以0.32μg-ml~(-1)的剂量诱发的突变频率较溶剂对照组高10.5倍，而B(α)P以1.1μg·ml~(-1)的剂量诱发突变频率较溶剂对照组高12倍，上述试验结果表明，已建立的系统检测已知阳性诱变物结果明显，该系统可用于诱变检测。
质粒经6.6μg·ml~(-1)的喹乙醇处理后，转染Vero细胞，并从细胞中回收质粒转化E.coliMBM7070，试验结果表明，喹乙醇处理组的突变频率明显高于溶剂对照组。本研究又对突变子质粒靶基因进行序列测定，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。
质粒经1.90、3.75、7.5、15、30μg·ml~(-1)的喹烯酮处理后，将其转染Vero细胞，回收并转化E.coli MBM7070，试验结果表明，喹烯酮处理组的突变子数较溶剂对照没有明显的增加；试验又采用S_9将相同浓度的喹烯酮代谢活化后处理质粒，转染Vero细胞，经回收并转化E.coli MBM7070进行突变子筛选，试验结果表明，喹烯酬处理组的突变子数较阴性对照仍然没有明显增加，喹烯酮诱变检测结果阴性。
A SV40 based shuttle vector pSP189 and African green monkey kidney cell line (Vero) was appied , which constitute a shuttle vector /mammalian cell system to detect mutagenesis in vitro induced by animal drugs olaqiundox and quinocetone. In the study, plasmids pSP189 were transfected into vero cells by positive ion LipofectMINA2000 reagent. A fter replicating in the mammalian cells,and then picked up and returned to E.coli MBM7070 for analyzing transformants and mutatants. The spontaneous mutation frequency was 1.9×10-4 in pSP189/vero.In order to further confirm that pSP189/vero could be utilized for detection of mutagenicity ,known mutagen MNNG and B(a)P were used to treat pSP189/vero.Results showed that the mutation frequency induced by 0.32ug.ml-1 and 1.1ug.ml-1 B(a)P were about 10.5 folds and 9 folds higher than that of the background lever.
The leision induced by 6.6ug.ml-1olaqiunxox included a large number of basesubstitutions,in addition to complex deletions. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background lever.
However the number of mutation induced by 1.90,3.75, 7.5, 15, 30ug.ml-1 qiuncetone had no obvious changes over the spontaneous background.Rat-liver microsomes were used to activate quinocetone for in vitro modification of the pSP189 shuttle vector.Modified plasmids were transfected into Vero cells,then recovered and transfected into E.coli MBM7070 for mutant identification.But in the reseach can not find mutations increasing over the spontaneous background lever, so qiuncetone can not: effect plasmids DNA mutate,
质粒经6.6μg·ml~(-1)的喹乙醇处理后，转染Vero细胞，并从细胞中回收质粒转化E.coliMBM7070，试验结果表明，喹乙醇处理组的突变频率明显高于溶剂对照组。本研究又对突变子质粒靶基因进行序列测定，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。
质粒经1.90、3.75、7.5、15、30μg·ml~(-1)的喹烯酮处理后，将其转染Vero细胞，回收并转化E.coli MBM7070，试验结果表明，喹烯酮处理组的突变子数较溶剂对照没有明显的增加；试验又采用S_9将相同浓度的喹烯酮代谢活化后处理质粒，转染Vero细胞，经回收并转化E.coli MBM7070进行突变子筛选，试验结果表明，喹烯酬处理组的突变子数较阴性对照仍然没有明显增加，喹烯酮诱变检测结果阴性。
A SV40 based shuttle vector pSP189 and African green monkey kidney cell line (Vero) was appied , which constitute a shuttle vector /mammalian cell system to detect mutagenesis in vitro induced by animal drugs olaqiundox and quinocetone. In the study, plasmids pSP189 were transfected into vero cells by positive ion LipofectMINA2000 reagent. A fter replicating in the mammalian cells,and then picked up and returned to E.coli MBM7070 for analyzing transformants and mutatants. The spontaneous mutation frequency was 1.9×10-4 in pSP189/vero.In order to further confirm that pSP189/vero could be utilized for detection of mutagenicity ,known mutagen MNNG and B(a)P were used to treat pSP189/vero.Results showed that the mutation frequency induced by 0.32ug.ml-1 and 1.1ug.ml-1 B(a)P were about 10.5 folds and 9 folds higher than that of the background lever.
The leision induced by 6.6ug.ml-1olaqiunxox included a large number of basesubstitutions,in addition to complex deletions. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background lever.
However the number of mutation induced by 1.90,3.75, 7.5, 15, 30ug.ml-1 qiuncetone had no obvious changes over the spontaneous background.Rat-liver microsomes were used to activate quinocetone for in vitro modification of the pSP189 shuttle vector.Modified plasmids were transfected into Vero cells,then recovered and transfected into E.coli MBM7070 for mutant identification.But in the reseach can not find mutations increasing over the spontaneous background lever, so qiuncetone can not: effect plasmids DNA mutate,
引文
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