Basic study for dunction and gene regulation of human fetal liver P-450

人胎肝P-450功能及基因调控的基础研究

基本信息

批准号：
02454482
负责人：
KAMATAKI Tetsuya
金额：
$ 3.9万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

1.A pM56 expression vector, which was ligated with P-450IIIA7 cDNA downstream of SRalpha promoter, was constructed. The pM56 was introduced into mammary tumor cell line, MCF-7, two transformants, M21 and M27 were obtained. We examined whether or not these transformants were capable of activating aflatoxin B_1, and lead to the cell death. The 50% lethal dose of aflatoxin B_1 for MCF-7 was 17.0 mug/ml, while that for M21 and M27 was 1.4 mug/ml. This result indicated that M21 and M27 became thirtween times more sensitive to aflatoxin B_1 than MCF-7. It was suggested that P-450IIIA7 protein expressed in M21 and M27 activated the the micotoxin to result in the cell death.2.We tried to express P-450lllA7 protein in an insect cell to obtain a larger amounts of P-450IIIA7 protein. The construction procedure was as follows ; P-450IIIA7 cDNA was inserted to the polyhedorin gene of nuclear polyhedorosis virus (NPV), and recombinant Virus (NPVHP1) was prepared. The insect cell (Sf9 cell) was infected with the NPVHP1. Then, the whole cell extracts from the infected Sf9 cells were prepared. One miligram of the whole extracts contained 1.10 nmol P-450IIIA7 protein. The umu test using the cell lysates showed that P-450IIA7 expressed in Sf9 cells converted aflatoxin B_1 to a mutagen.3.P-450IIIA4 (adult form) and P-450IIIA7 (fetal form) genes were isolated from human genomic library, and the sequences analyzed. Both genes had 13 exons over 20kb. Moreover, 5'flanking sequences between both genes had high similarity (91%).4.We isolated cDNAs of mouse P-450IIIA, which were termed as P-450IIIA_<M1> (P-450IIIA11) and P-450IIIA_<M2>, from liver cDNA library of ddY strain mouse. P-450IIIA_<M1> (P-450IIIA11) and P-450IIIA_<M2>, from liver cDNA library of ddY strain mouse. P-450IIIA_<M1> was constitutively expressed in mouse livers and induced by dexamethasone, while P-450IIIA_<M2> was expressed at a very low level.

1. A PM56表达载体与Sralpha启动子下游的P-450IIIA7 cDNA连接。将PM56引入乳腺肿瘤细胞系MCF-7，两个转化体M21和M27中。我们检查了这些转化体是否能够激活黄曲霉毒素B_1并导致细胞死亡。 MCF-7的50％致命剂量的黄曲霉毒素B_1为17.0毫克/mL，而M21和M27的致死剂量为1.4毫克/ml。该结果表明，M21和M27的时间比MCF-7对黄曲霉毒素B_1更敏感。有人提出，在M21和M27中表达的P-450IIIA7蛋白激活了微毒素，导致细胞死亡。2。我们试图在昆虫细胞中表达P-450LLLA7蛋白，以获得大量的P-450IIIIA7蛋白。施工程序如下；将P-450IIIA7 cDNA插入核多面毒病毒（NPV）的多面体基因，并制备重组病毒（NPVHP1）。昆虫细胞（SF9细胞）被NPVHP1感染。然后，制备了从感染的SF9细胞中的整个细胞提取物。整个提取物中的一个媒体含有1.10 nmol P-450IIIA7蛋白。使用细胞裂解液的UMU检测表明，在SF9细胞中表达的P-450IIA7将黄脂蛋白B_1转化为诱变剂。3.p-450IIIA4（成人形式）（成人形式）和P-450IIIA7（胎儿形式）基因与人基因组文库中分离出来，并分析了分析的序列。这两个基因的外显子超过20kb。此外，两个基因之间的5'Franking序列具有很高的相似性（91％）。4。我们分离的小鼠P-450IIIA的cDNA被称为P-450IIIA_ <M1>（P-450IIIIA11）和P-450IIIIA11）和P-450IIIIA__ <m2>，来自DDY菌株库的Liver CDNA库。 P-450IIIA_ <M1>（P-450IIIA11）和P-450IIIA_ <M2>，来自DDY菌株小鼠的肝cDNA库。 P-450IIIA__ <m1>在小鼠肝脏中组成型表达并由地塞米松诱导，而P-450IIIA__ <m2>在非常低的水平上表达。