【摘要】 目的制备兔抗人细胞色素P450酶1A2、3A4及4A11的特异性抗体,对其在药物代谢方面的应用进行研究。方法①利用生物信息学方法分析CYP1A2、CYP3A4及CYP4A11蛋白的序列,利用DNAstar软件中蛋白质的亲水性结构、柔韧区、抗原指数及表面概率结构指标选择多肽序列,应用Blast软件对所选序列进行同源性分析并合成多肽,与载体蛋白钥孔戚血蓝素(KLH)偶联,免疫大耳白兔制备抗CYP1A2、3A4及4A11抗体。抗体纯化后经酶联免疫吸附(ELISA)、免疫印迹(Western blot)鉴定其特异性;②通过制备大鼠给药模型利用Western blot评价上述抗体在药物代谢酶分析方面的作用。结果获得抗CYP1A2、CYP3A4及CYP4A11合成肽的抗体。Western blot结果显示,三种抗体均可与其合成肽及天然CYP1A2、CYP3A4及CYP4A11蛋白出现特异性反应,识别的相应抗原的相对分子质量(Mr)分别为63 000、59 000及53 000。同时,Western blot结果显示,所制备的抗体能识别药物诱导后大鼠天然CYP1A2、CYP3A4及CYP4A11。结论合成的多肽-KLH复合物具有免疫原性,可用于制备相应的抗体。制备的抗体特异性良好,可应用于药物代谢酶方面的研究。更多还原

【Abstract】 Objective To generate and investigate rabbit polyclonal antibodies against human peptides of CYP450 enzymes.Methods ①Human peptides of CYP450 enzymes and their proteinic sequence were analyzed by means of bioinformatics,and certain sequences were chosen and synthesized according to four indexes(Hydrophilicity Plot,Flexible Regions,Antigenic Index and Surface Probability Plot) after homology analysis by software Blast.The synthesized peptides were conjugated to keyhole limpet hemocyanin(KLH) to increase antigenicity.Rabbits were immunized with KLH conjugated peptides.The polyclonal antibodies induced were charactered by ELISA and Western blot;②The polyclonal antibodies were tested by Western blot in drug metabolism of rat model.Results Three kinds of polyclonal antibodies against CYP450 enzymes were generated successfully.Western blot results showed that the antibodies recognized proteins with molecular weight of 63 000,59 000 and 53 000,respectively,the antibodies could specifically react with synthesized peptides of CYP450 enzymes as well as natural human liver microsomal proteins.Western blot results also showed that the antibodies can discern the changes of natural CYP1A2,CYP3A4 and CYP4A11 of rats induced by drugs.Conclusion The synthesized CYP450 peptides can be used to immunized rabbit in order to generate polyclonal antibodies against human CYP450.The antibodies prepared can be used as potential tools to study roles of CYP1A2,CYP3A4 and CYP4A11 in drug metabolism.更多还原