兔抗人细胞色素P450 1A2多克隆抗体的制备及鉴定

Generation and characterization of rabbit polyclonal antibody against peptides of human CYP1A2

目的：制备兔抗人细胞色素P4501A2（CYP1A2）抗体及其特性鉴定。方法：利用生物信息学方法分析CYP1A2蛋白的序列，根据亲水性、抗原性、柔韧性及表面性等指标选择多肽序列，合成CYP1A2多肽，与载体蛋白钥孔戚血蓝素（Keyhole limpet hemocyanin，KLH）偶联，免疫日本大耳白兔制备抗CYP1A2抗体。辛酸-硫酸铵法纯化抗体，间接ELISA法测定抗体的效价及相对亲和力常数，Western blot鉴定其特异性，免疫组化进行定位。结果：获得针对小分子CYP1A2的抗体。该抗体效价为1∶16000；相对亲和力常数在10^5-10^6/M，具有实用价值；可与CYP1A2合成肽及天然CYP1A2出现特异性反应；可识别正常人肝脏组织CYP1A2；Western blot的结果显示，该抗体识别的相应抗原的相对分子质量为58000。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。制备的兔抗CYP1A2合成肽抗体可应用于酶联免疫吸附试验、免疫印迹、免疫组化实验。

Objective：To generate and identify the rabbit polyclonal antibody against peptides of human CYP1A2. Methods： The peptide sequences of human CYP1 A2 were analyzed according to its bioinformatics, and synthesized on the basis of hydrophilicity, antigenicity, accessibility and flexsibihty. The synthesized peptides were cross-linked with keyhole limpet hemocyanin（KLH） , which was used to immunize rabbits for production of Ab. The polyclonal antibodies purified by octoic-ammonium sulfate were characterized by ELISA, Western blot and immunohistochemistry （IHC）. Results： The polyclonal antibodies against KLH conjugated CYP1 A2 poptides were prepared successfully ,with the titer of 1：16 000. Western blot identified that the antibodies that recognize protein with molecular weight of 58 000 could specifically react with synthesized peptides of CYP1 A2 and the natural microsome CYP1 A2 of human liver. IHC results showed that the antibodies could recognize microsome CYP1 A2 in the human liver tissue. Conclusion ：The synthesized peptides can be used to immunize rabbits to generate polyclonal antibodies. The antibodies prepared can be used as a potent tool in the studies on CYP1A2.