The cloning of ESAT-6, EspC and Rv2029c genes obtained from an Indonesian tuberculosis-infected patient

• Published in: IOP conference series. Earth and environmental science 2023-12, Vol.1271 (1), p.12091
• Main Authors: Hidayah, N, Pambudi, S, Widayanti, T, Suryanggono, J, Luqman, M, Mulyawati, L, Nurlaila, I, Novianti, T, Nie, N
• Format: Article
• Language: English
• Subjects: Ampicillin; Antigens; Biomarkers; Cloning; E coli; ESAT-6; ESAT-6 antigen; Escherichia coli; EspC; Genes; Genomes; Polymerase chain reaction; Proteins; Rv2029c; Sputum; Tuberculosis; Vaccines; Virulence
• ISSN: 1755-1307; 1755-1315
• DOI: 10.1088/1755-1315/1271/1/012091

Early secreted antigenic target of 6-kDa protein (ESAT-6), a protein related to tuberculosis virulence (EspC) and latent antigen Rv2029c are important biomarkers for Mycobacterium tuberculosis prevention. Recent advances in subunit protein vaccines offer promising solutions to the inadequacies of existing BCG vaccines. In this study, these antigens were cloned into pET plasmid series to express the genes using the Escherichia coli expression system. Polymerase Chain Reaction was used to amplify the target genes from genome DNA isolated from Indonesia tuberculosis-infected patient’s sputum. A 309 bp of ESAT-6 fragment and a 337 bp of EspC fragment were inserted into pET-21b. Meanwhile, the 1038 bp of Rv2029c fragment was inserted into pET-32b. All constructs were transformed into Escherichia coli TOP10 and screened on Luria Bertani agar media containing ampicillin. The transformants were subjected to colony PCR using T7 universal primers followed by plasmid isolation to confirm the recombination. All genes were successfully cloned into the pET plasmid series. These results serve as a basis for further studies to express the recombinant protein as a booster vaccine.