穿梭质粒pSP189/哺乳动物细胞系统在AFB_1诱变分子机理研究中的应用  

Molecular Mechanism of Mutagenesis Induced by Aflatoxin B 1 Using a Shuttle Vector pSP189/Mammalian Cell System

作　　者：谈幸之[1] 李申德[1] 罗雪云[1] 周宇红[1] 冯晓莲[1] 杨晓洁 崔惠云 

机构地区：[1]卫生部食品卫生监督检验所

出　　处：《卫生研究》1997年第4期271-277,共7页Journal of Hygiene Research

基　　金：国家自然科学基金;卫生部青年基金

摘　　要：ＡｆｌａｔｏｘｉｎＢ１（ＡＦＢ１）是食品中广泛存在的致癌性最强的霉菌毒素之一，本文将基于ＳＶ４０病毒的短暂复制型穿梭质粒ｐＳＰ１８９与非洲绿猴肾细胞（ＶｅｒｏＥ６细胞系）组成穿梭质粒／哺乳动物细胞诱变检测系统检测ＡＦＢ１的诱变性，并通过对靶基因ＳｕｐＦＴＲＮＡ的序列分析了解ＡＦＢ１在ＤＮＡ一级结构上的作用位点，类型及序列特异性等信息。结果表明：在体外采用大鼠肝微粒体与ＡＦＢ１和ｐＳＰ１８９直接作用形成ＡＦＢ１－ＤＮＡ加合物，再转染ＶｅｒｏＥ６细胞，随着ＡＦＢ１作用质粒ＤＮＡ时间的延长，经哺乳动物细胞内复制并在宿主菌中筛选得到的突变体逐渐增加，并呈明显的剂量－反应关系，检出的突变体经０．８％琼脂糖凝胶电泳分析大多为点突变，对其中５３个独立突变子的靶基因ＳｕｐＦＴＲＮＡ的序列分析结果表明：ＡＦＢ１诱发ｐＳＰ１８９靶基因ＳｕｐＴＩＲＮＡ的突变大多单碱基置换，占突变子总数的８４．９％，９５．２％的碱基置换发生在ＧＣ位点，其中以ＧＣ→ＴＡ的颠换占大多数，约为５３．３％，其次为ＧＣ→ＡＴ的转换，约为３５．６％；ＡＦＢ１诱发的突变在靶基因ＳｕｐＦＴＲＮＡ的分布并非是随机的，而是存在着一定的序列特异性，其特征序列为“５＇－?AFB 1 is one of the most potent carcinogenic mycotoxins, naturally occurred in foods. We applied a new SV40 based shuttle vector pSP189 and African Green kidney cells (VeroE6 cell line), which constitute shuttle vector/mammalian cell system to detect mutagenesis induced by AFB 1 and to study the effects of AFB 1 on the mutation site, type and sequence specificity of DNA molecular level through sequence analysis of pSP189 target gene SupF TRNA. The results indicated that through various time treatment of AFB 1 and rat liver microsome, the mutants were obtained by transformation of E.Coli MBM 7070 with progeny of pSP189 generated during replication in veroE6 cells, which increased gradually with the lapse of time, and this experiment showed a significant dose response relationship. Most detected mutants were point mutations evidenced by agarose gel electrophoresis analysis. The results of direct sequencing SupF TRNA of 53 independent mutants showed that most mutations (about 84 9%) were single base substitutions, among which 95.2% of base substitutions happened on the site of G∶C base pair, the predominant mutation was G∶C→T∶A transversion, accounting for 53.3%, followed by G∶C→A∶T transition, accounting for 35.6%. AFB 1 induced mutations did not distributed randomly, and had mutation hot spots and sequence specificity, which contained 5 base pair sequence 5' NNTTC 3'. The mutations on the SupF shuttle vector were consistent with the results from studies of oncogene tumor suppressor genes and DNA adduct.

关 键 词：穿梭质粒 黄曲霉毒素 诱变 癌变 

分 类 号：R730.231.3[医药卫生—肿瘤]