Construction and Identification of Mycobacterium tuberculosis Mtb8.4 Eukaryotic Expression Plasmid and Prokaryotic Expression Plasmids

The gene encoding the Mtb8.4 protein was amplified based on the genome DNA sequence of H37Rv by PCR and was then cloned into eukaryotic expression vector pJW4303,prokaryotic expression vector pGEX respectively after digesting with restriction endonuclease corresponding to the enzyme sites on the vectors.These recombinant plasmids were identified by means of restricted enzyme digestion, PCR and DNA sequence analysis. Results showed that these plasmids were successfully constructed and the accuracy of these constructs was confirmed. Finally, the prokaryotic recombinant plasmid was transferred into E.coli BL21(DE3)plysS and induced by IPTG to express the protein. This method could be used for the diagnosis of tuberculosis, preparation of subunit vaccine and DNA vaccine and application of those vaccines.