人丙氨酸氨基转移酶(ALT2)的表达、单克隆抗体制备及鉴定

Expression of recombinant human alanine aminotransferase(ALT) isoenzyme 2 and preparation of anti-ALT2 monoclonal antibody

目的:克隆、表达、纯化重组人丙氨酸氨基转移酶(ALT2),以此作为抗原免疫动物获得针对ALT2的单克隆抗体(mAb)株,用于ALT的免疫诊断。方法:通过RT-PCR从肝癌细胞中扩增丙氨酸氨基转移酶(ALT2)基因,并将其克隆至pET-28a表达载体中,带有组氨酸标签的ALT2蛋白经镍亲和层析纯化,纯化的ALT2作为抗原免疫小鼠制备mAb,通过Western blot和ELISA方法测定mAb的亲和力和特异性。结果:利用大肠杆菌成功表达ALT2蛋白,以镍亲和层析纯化获得ALT活性超过10 000 U/L的ALT2目的蛋白,以此蛋白免疫小鼠后得到5株mAb。结论:经过初步筛选获得2株高特异性的mAb,为研制ALT2蛋白定量检测试剂提供了重要工具。

AIM： To clone and express human alanine aminotransferase 2（ALT2） in E.coli Rosetta（DE3）,and to prepare monoclonal antibodies（mAb） against ALT2 for diagnostic purpose.METHODS： The gene encoding alanine aminotransferase 2（ALT2） was cloned from hepatoma carcinoma cell by RT-PCR,and then inserted into pET28a vector.Recombination plasmids（pET28a-ALT2） were transformed into E.coli BL21.Human ALT2 was expressed as His-tagged fusion proteins and purified by immobilized Ni2＋-affinity chromatography.The purified fusion ALT2 protein was used as an antigen to prepare mAb against it.RESULTS： The fusion ALT2 protein was expressed in recombinant E.coli Rosetta（DE3）.The enzymatic activity of purified His-tag ALT2 is over 10 000 U/L.Mice were immunized with the purified fusion ALT2 protein,and 5 mAbs against ALT2 were generated.CONCLUSION： Two mAbs with high specificity for ALT2 were selected for further quantitative diagnostic reagent development.