Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphi...Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphilis vaccine. Method: The Tpp17 lipoprotein gene was amplified fromthe TP(strain Nichols), and then it was recombinated into aplasmid pMAL-2c and cloned within E coli l2-TB1. The hostbacteria containing recombinant plasmids were induced withIPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double diges-tion and PCR. Recombinant plasmids were transformed intoE. coli and the E coli carrying recombinant plasmids wereinduced. The expression of TP 17KD was detected by sodiumdedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showedthat the induced E coli carrying recombinant plasmid couldproduce 60KD fusion protein at high levels. Gel scanningshowed that 17KD protein expression in E coli accounted for10% of total cellular protein. The recombinant protein antigenreacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developingnew techniques of laboratory diagnosis for syphilis and newvaccines. Preliminary clinical application showed that thefusion protein could be used for the diagnosis of syphilis.展开更多
文摘Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphilis vaccine. Method: The Tpp17 lipoprotein gene was amplified fromthe TP(strain Nichols), and then it was recombinated into aplasmid pMAL-2c and cloned within E coli l2-TB1. The hostbacteria containing recombinant plasmids were induced withIPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double diges-tion and PCR. Recombinant plasmids were transformed intoE. coli and the E coli carrying recombinant plasmids wereinduced. The expression of TP 17KD was detected by sodiumdedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showedthat the induced E coli carrying recombinant plasmid couldproduce 60KD fusion protein at high levels. Gel scanningshowed that 17KD protein expression in E coli accounted for10% of total cellular protein. The recombinant protein antigenreacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developingnew techniques of laboratory diagnosis for syphilis and newvaccines. Preliminary clinical application showed that thefusion protein could be used for the diagnosis of syphilis.
