人细胞色素P450氧化还原酶的克隆表达和抗体的制备

Cloning and Expression of Human Cytochrome P450 Reductase and Production of Polyclonal Antibodies Against the Recombinant Protein

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.

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 pT7450 from pET-21 a （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 cytoehrome P450s.