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

1.构建了连接SRalpha启动子下游P-450IIIA7 cDNA的pM56表达载体。将pM56导入乳腺肿瘤细胞系MCF-7，获得M21和M27两个转化子。我们检测了这些转化子是否能够激活黄曲霉毒素B_1并导致细胞死亡。MCF-7的50%致死量为17.0马克/毫升，M21和M27的50%致死量为1.4马克/毫升。结果表明，M21和M27对黄曲霉毒素B_1的敏感性是MCF-7的13倍。提示M21和M27中表达的P-450IIIA7蛋白激活微毒素导致细胞死亡。我们尝试在昆虫细胞中表达P-450lllA7蛋白，以获得更大量的P-450IIIA7蛋白。施工流程如下：将P-450IIIA7 cDNA插入核型多角体病毒（NPV）多角体蛋白基因，制备重组病毒（NPVHP1）。用NPVHP1感染昆虫细胞（Sf9细胞）。然后，从感染的Sf9细胞中制备全细胞提取物。每毫克全提取物含有1.10 nmol P-450IIIA7蛋白。利用细胞裂解物进行的umu试验表明，在Sf9细胞中表达的P-450IIA7可将黄曲霉毒素B_1转化为诱变物。从人类基因组文库中分离P-450IIIA4（成虫型）和P-450IIIA7（胎儿型）基因，并对其序列进行分析。两个基因都有13个超过20kb的外显子。此外，两基因间的5'侧链序列具有较高的相似性（91%）。从小鼠ddY品系肝脏cDNA文库中分离到小鼠P-450IIIA的cDNA，分别命名为P-450IIIA_<M1> (P-450IIIA11)和P-450IIIA_<M2>。P-450IIIA_<M1> (P-450IIIA11)和P-450IIIA_<M2>，来自ddY品系小鼠肝脏cDNA文库。在地塞米松诱导下，P-450IIIA_<M1>在小鼠肝脏中组成性表达，而P-450IIIA_<M2>表达水平极低。

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.