人类肝脏细胞色素P-450 4F新基因的分离与表达

目的 从人类胎肝ｃＤＮＡ基因文库中分离细胞色素Ｐ－４５０４Ｆ新基因并在体外表达。方法 用聚合酶链式反应（ＰＣＲ）方法从人类胎肝ＣＤＮＡ基因文库分离中全长ｃＤＮＡ克 ，用ＤＮＡ自动测序仪双方测序，体外酵经平细胞中表达蛋白质。结果 ＤＮＡ序列分析示ＣＤＮＡ全长１９８０碱基对，编码一个５２０个氨基酸的蛋白质，相对分子量５９７００，ｃＤＮＡ编码的氨基酸序列与人类白细胞的白三烯Ｂ４Ｗ－羟化酶不（ＣＹＰ４Ｆ３

Isolation and sequencing of a novel form of cytochrome p-450 4F family from human liver

Objective To isolate and sequence a novel gene of cytochrom p-450 4F family. Methods A cDNA was isolated by touchdown PCR from a cDNA library of human fetal liver using specific primers. The DNA sequencing was done by an auto-sequence machine through forward and reverse directions. The expression of cDNA encoded protein was tested in yeast cells. Results Sequencing of the full lenghth cDNA revealed a sequence of 1980 nucleotides coding for a protein of 520 amino acids with a molecular weight of 59 700 Da. The cDNAdeduced amino acid sequence showed 92% homology to that of human polymorphonuclear leukocyte (PMN) LTB4-hydroxyase (CYP4F3) and 91% homology to that of human liver LTB4 hydroxylase (CYP4F2). The expression of protein in the yeast cells showed a fusion protein with a molecular weight of about 86 000 Da and 60 000 Da after a proteolytic removal of the GST affinity tag. It was confirmed by RT-PCR that the gene was expressed in the adult liver. Conclusion Our results clearly show a novel form of cytochrome p-450 4F in human liver. (GenBank accession number AF054821 ).