以重组P-gp为抗原建立检测MDR 1抗体间接ELISA方法的研究

目的:构建MDR 1基因原核表达质粒，表达P-gp重组蛋白，建立检测MDR1抗体的间接ELISA方法。方法:利用重组PCR技术扩增MDR 1基因的1kb片段，克隆至pET-28b（＋）中，构建原核表达质粒pETP-gp，转染感受态菌BL21（DE3）和BL21（DE3）plyss；以E.coli高效表达的P-gp基因主要抗原编码区重组蛋白为抗原，以HRP标记的兔抗人IgG为二抗，建立间接ELISA检测方法。结果:正确构建了pETP-gp原核表达质粒，并可在E.coli中高效表达，表达蛋白可用作检测MDR 1抗体ELISA抗原。结论:成功表达出重组蛋白P-gp，建立了检测MDR 1抗体的间接ELISA方法。

Indirect ELISA for the detection of serum antibodies against MDR 1with the E.coli high expressed recombinant pETP-gp protein as antigen

The main reason of Multidrug resistence is P-gp overexpressed in vivio. Because of its expression and localization, it has been suggested that P-gp plays an important role in cancer chemotherapy. In this study, we performed a method for monitoring P-gp overexpressed. A recombinant plasmid named pETP-gp was constructed by cloning MDR 1 1kb gene into prokaryotic expression vector pET-28b(+). E.coli competent host BL21(DE3) and BL21(DE)lyss were transformed with pETP-gp. The recombinant protein P-gp was high level expression in the two Ecoli BL21(DE3) and BL21(DE)lyss after induced by IPTG. Recombinant proteins were used as detecting antigen, and then the optimal reactive condition and the concentration of the second antibodies were confirmed. The expression in BL21(DE3)plyss was higher until to 48% of the total protein of the induced recombinant bacteria. Expression protein was shown as specificity P-gp by immunoprint. It was the first report that MDR 1 1kb gene product was expressed with high level in E.coli. Indirect ELISA for the detection of serum antibodies against MDR 1 was established with the E.coli high expressed recombinant pETP-gp protein as antigen and the rabbit against human IgG by tagging HRP as the second antibodies.