建立pESnx／FL穿梭质粒致突变测试系统的实验研究

本研究用电穿孔法将带有邻苯二酚氧化酶基因（ｘＹ１Ｅ基因）的质粒ｐＥＳｎｘ转染至５种哺乳动物细胞（ＦＬ，ＨＴ，ＬＴＲ，ＰＡ３１７和Ｗｇ３－ｈ）。结果表明，质粒ｐＥＳｎｘ在５种细胞中均可形成抗性克隆。但是，仅ｐＥＳｎｘ／ＦＬ克隆可获得每皿５０个以上的转化子和低于１０＾４的自发突变率，符合穿梭质粒测试系统的要求。为进一步验证可用于检测致突变性，用已知可诱发点突变的致癌物亚硝基胍（ＭＮＮＧ）处理ｐＥＳｎｘ

Experimental Studies on the Development of pESnx/FL Shuttle Vector Plasmid Mutation Test System

In this study, plasmid pESnx with xY1E gene(catecholgene)were transfected into 5 kinds of mammalian cells(FL, HT, LTR, PA317,and Wg3-h)by electroporation. Results showed that plasmid pESnx could form resistant colonies in all of the five mammalian cells, but transformants could be produced more than 50/amp plate in pESnx/FL only when it was transfected into MC1061F'Kan. The spontaneous mutation frequency was less than 10-4 in pESnx/FL. Therefore, pESnx/FL accord with the requirements of shuttle vector plasmid mutation test system. In order to further confirm that pESnx/FL could be utilized for detection of mutagenicity, a known mutagen MNNG which could induce point mutation was used to treat pESnx/FL colonies. Results showed that the mutation frequency induced by MNNG was 27.8 folds higher than that of the spontaneous mutation frequency and had dose-effect dependent. The mutants were identified by Cracking gel rapid extraction. Results revealed that the shuttle vector was stable, there was no large deletion of DNA and change of molecular weight, and it was verified that mutation induced by MNNG was a point mutation.