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《Journal of Hebei University(Natural Science Edition)》 2006-01
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Cloning and Expression of Human Cytochrome P450 Reductase and Production of Polyclonal Antibodies Against the Recombinant Protein

CHENG Jie~(1,2),GONG Yi~3,CHEN Yue~3,YANG Ling~1(1.Laboratory of pharmaceutical resource discovery,CAS.,Dalian 116023,China;2.Graduate School of Chemical Academy of Science,Dalian 116023, China;3.Shanghai Research Center of Bioengineering,Chinese Academy of Sciences,Shanghai 200233,China)  
Cytochrome P450 reductase(CPR) is a diflavin enzyme responsible for electron donation to mammalian cytochrome P450 enzymes which makes a key role in exerting the activity of cytochrome P450s.In this article,a recombinant cDNA of human placental NADPH-cytochrome P-450 reductase(hCPR),cut off from the plasmid pCMV-hCPR,was ligated into a reconstructed vector pT-7450 from pET-21a (Novagen).The hCPR protein expressed in Escherichia coli BL21(DE3) was recovered into the inclusion body of cell lysate and purified to homogeneity by affinity chromatography on Ni sepharose 4B.After purification,about 49 mg hCPR was obtained from one liter of fermentation ligour,and equivalent to about 10% of the total protein.Spectra assay certificated the NADPH-dependent cytochrome c reducing activity 65 u/mg.Purified rabbit polyclonal antibodies were produced against recombinant hCPR and the value and specialty of the antibodies were analyzed by an enzyme-linked immunosorbent assay(ELISA),the result show the value reaches to 1∶200,00.The recombinant protein of hCPR and the polyclonal antibodies could be applied for study of drug metabolism in vitro and functional mechanism between CPR and cytochrome P450s.
【Fund】: 国家863自然科学基金资助项目(2003AA223061)
【CateGory Index】: Q78
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