Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis

Surface display of multi-epitopes synthetic gene of Mycobacterium tuberculosis on Salmonella typhi Ty21a using a pgsA anchor protein

“…The pVaxVacIII contains the VacIII gene which was constructed using the assembly PCR technique. VacIII combines a ubiquitin moiety (UbGR) and several T-cell epitopes of mycobacterial proteins genes: ESAT6 (P1), ESAT6 (P2), PhoS1 (38kD)(129-137), PhoS1 (38kD)(65-83), PhoS1 (P3), Hsp 16.3 and MTB 8.4 genes (Fang et al, 2006;Fang et al, 2007). Based on the immunogenicity study in mice, pVaxVacIII stimulate strong Th1 cell response as characterized by high IFNproduction and low IL-4 production (Fang et al, 2007).…”

“…This expression system requires specific conditions to express the recombinant protein namely IPTG induction and an incubation temperature of 25°C for optimal expression (Lee et al, 2000;Fang et al, 2006). Since the oral route is used, the vaccine candidate will be introduced into an unfavorable environment for antigenic protein expression after ingestion.…”

“…This plasmid contains the HCE promoter and pgsA surface display system. VacIII gene was isolated from pTInak-VacIII (Fang et al, 2006).…”

“…Synthetic MTB 8.4 peptides (Table 1) were synthesized by Genemed Synthesis, Inc. (CA, USA). Recombinant VacIII protein was kindly given by Dr. Fang Chee Mun (Fang et al, 2006).…”

“…The VacIII is a multi-epitope synthetic gene, constructed by assembly PCR, which consists of UbGR (Ubiquitin) and seven T-cell epitopes of MTB proteins: ESAT-6 (P1), ESAT-6 (P2), Phos1 (65-83), Phos1 (129-137), Phos1 (166-175), Hsp 16.3 and Mtb 8.4) (Fang et al, 2006).…”