摘要
Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.
Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.
基金
ThisworkwassupportedbygrantsfromtheNational?63?HighTechnologyProgramofChina(No.2001AA221031)
theNationalNaturalScienceFoundationofChina(No.30300383).
