Expressing Nitroreductase(NTR)in E.coli And Preparing Its Antibody

In this study, using a single EcoRI cut prebuilt plasmid containing the NTR gene T-easy/NTR will cut out the NTR gene fragment was cloned into the prokaryotic expression vector pRSET B in, and the NTR gene sequencing, has been NTRthe correct sequence, EcoRI-cut and single HindIII / XbaI double digestion and sequencing were correctly identified.While establishing the E. coli expression system, and NTR-pRSET B/BL21 IPTG induced expression of the relative molecular mass (M r) of approximately 27kDa soluble fusion protein.Tested and the same need to express the NTR protein, explained that it had successfully completed the extraction and E. coli NTR gene engineering bacteria to build, and its expression in the process were studied.The process of expressing a preliminary study, the basic parameters have been expressed.Purification process after the selection of several reagents used Ni +-NTA affinity Precipitation of soluble proteins obtained after purification.SDS-PAGE showing the purification process of the detection process has reached the electrophoretic purity, NTR protein purity has been tested to meet the need.Experiments using this isolated NTR protein immunization of rabbits in order to get NTR protein antibodies, test results show that the result of the immune rabbits is very good.Western Blot been immune rabbit antibodies specifically recognized the fusion protein, the antibody can detect both the eukaryotic expression of NTR, as the late NTR protein expression in eukaryotes suicide gene detection and determination of the course are laidbasis.Western blot showed that prokaryotic expression of NTR fusion protein with a good immunological activity, self-made anti-rabbit serum for the NTR NTR gene in further studies of eukaryotic cells and for the treatment provides a useful reagent.