sars冠状病毒核衣壳蛋白对细胞色素氧化酶cyp450cyp4f3a)酶活性影响的研究-effect of nucleocapsid protein of sars coronavirus on cytochrome oxidase cyp 450 cyp 4f 3a ) enzyme activity.docx

ABSTRACTThe effects of SARS-CoV nucleocapsid protein on the catalytic activity ofCytochrome P450(CYP4F3A) AbstractNucleocapsid protein is one of the major structural proteins of SARS-CoV，Which is known to bind viral RNA to form the helical nucleocapsid. Also, Nucleocapsid protein of SARS-CoV is the major immunogenic antigen which was capable of generating strong humoral and cellular immune responses, suggesting an important application of SARS N protein in SARS diagnosis and prevention. The SARS N protein was shown to play important roles such as regulation of SARS-CoV RNA synthesis and nucleocapsid formation, disassembly of the viral core by binding to human cyclophilin A, and activation of AP-1 signal transduction pathway, which interferences host cell division and induces apoptosis in COS-1 cells in the absence of growth factors. Our previous work also demonstrated that the N protein must be importantly involved in the process of SARS-CoV infection.To delineate the molecular approach by which N protein was involved in the pathology, full length SARS- CoV N protein was employed as bait protein to screen a cDNA library from human fetal liver in the yeast two hybridization system. The results indicated that SARS-CoV N protein may interact with human Cytochrome P450 family 4 isoform 3 (CYP4F3). In this study, in vitro, the enzymatic reaction system and assay method of kinetic constant was established.We proved that the ω-hydroxylase activity of CYP4F3A can be significantly inhibited by binding with SARS-CoV N protein through a series of ion-change chromatography, molecular sieve gel filtration chromatography, immunoprecipitation, western blot and ELISA assay, and then degradation of the substrate(LTB4, an important inflammatory factor) was greatly suppressed. Furthermore, The Km values (Michaelis constant) of CYP4F3A determinated by Lineweaver-Burk plotting have changed from 2.5μmol/L to 10.2μmol/L, compared to the Km value of CYP4F3A(4.82μmol/L) when equivalence 229