Cloning, Expression and Purification of Espc, Espb and Espc/Espb Proteins of Mycobacterium tuberculosis ESX-1 Secretion System.

ABSTRACT: Background:It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB). The BCG vaccine is widely used to fight against TB; however, many question its ability to provide complete protection from Mtb. Recently, the "Region of Difference 1" (RD1) set of genes were shown to be involved in the pathogenesis of Mtb. Downstream of RD1 transcription region, two proteins are encoded, known as EspB and EspC, which were found to contribute to Mtb virulence.In this study these two proteins are targeted as potential vaccine candidates against TB. Methods:The EspB and EspC Mtb genes were codon-optimized for expression and synthesis in Escherichia coli (E. coli). The amplicons were cloned into a pET21a expression vector and transformed into E. coli BL21(DE3). The expression and purity of the expressed proteins (i.e. rEspC, rEspB and rEspC/EspB) were confirmed by SDS-PAGE and Western blotting. Moreover, BALB/c mice were immunized against Mtb using the recombinant proteins. Finally, the mice sera were analyzed via Western blotting. Results:EspC, EspB, and EspC/EspB fusion genes were cloned and expressed in E. coli. Both SDS-PAGE and Western blots confirmed the presence and successful purification of the desired proteins. Moreover, antisera produced against the purified recombinant proteins reacted with Mtb proteins. Conclusion:rEspC, rEspB, and rEspC/EspB could be expressed and purified using an E. coli expression system. The recombinant proteins induced the production of antibodies in BALB/c mice that reacted with Mtb proteins.

SUBMITTER: Salemi O 

PROVIDER: S-EPMC7275833 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature