[ Objective] In order to clone and express the formyltransferase of Brucella abortus in E. coli, purify the expressed protein and detect its immunogenicity. EMethods] A gene recoding formyltransferase 27 -35 ku （Wbkc...[ Objective] In order to clone and express the formyltransferase of Brucella abortus in E. coli, purify the expressed protein and detect its immunogenicity. EMethods] A gene recoding formyltransferase 27 -35 ku （Wbkc） was amplified from the genomic DNA of Brucella abortus PCR. The amplified fragments were digested with BamH I and Sal I, and then cloned to the vector pET28a. The constructed recombinant plasmid pET28a-Wbkc was transformed to E. coil BL21 and was induced to express the fusion protein. Then the protein was purified by histidine-binding res- in column chromatography, and the immunogenicity was detected by Western blot. The PCR product of Wbkc gene was cloned to the vector pET28a, and the recombinant vector was confirmed by colony PCR identification, recombinant vector digested identification and sequencing analy- sis. [ Results] It was successfully expressed in E. coil BL21 as a fusion protein with histidine at the presence of IPTG, and a specific protein band of 29 ku was found when identified by SDS-PAGE. Western blot showed good immunoreactivity of the expressed product. Wbkc was successfully cloned and expressed, and the purified fusion protein had immunogenicity. This study provides a solid foundation for the further study on the function of proteins and brucellosis diagnostic antigens. [ Conclusion] Amplification of formyltransferase gene of Brucella abortus and prokaryotic expression of WbkC_ indicatino the formvItransferase has specific immune reactivitv.展开更多
基金funded by the Natural Science Foundation o China(31060352)
文摘[ Objective] In order to clone and express the formyltransferase of Brucella abortus in E. coli, purify the expressed protein and detect its immunogenicity. EMethods] A gene recoding formyltransferase 27 -35 ku （Wbkc） was amplified from the genomic DNA of Brucella abortus PCR. The amplified fragments were digested with BamH I and Sal I, and then cloned to the vector pET28a. The constructed recombinant plasmid pET28a-Wbkc was transformed to E. coil BL21 and was induced to express the fusion protein. Then the protein was purified by histidine-binding res- in column chromatography, and the immunogenicity was detected by Western blot. The PCR product of Wbkc gene was cloned to the vector pET28a, and the recombinant vector was confirmed by colony PCR identification, recombinant vector digested identification and sequencing analy- sis. [ Results] It was successfully expressed in E. coil BL21 as a fusion protein with histidine at the presence of IPTG, and a specific protein band of 29 ku was found when identified by SDS-PAGE. Western blot showed good immunoreactivity of the expressed product. Wbkc was successfully cloned and expressed, and the purified fusion protein had immunogenicity. This study provides a solid foundation for the further study on the function of proteins and brucellosis diagnostic antigens. [ Conclusion] Amplification of formyltransferase gene of Brucella abortus and prokaryotic expression of WbkC_ indicatino the formvItransferase has specific immune reactivitv.
