Cloning, expression and purification of a gene encoding CFP-10 and ESAT-6 protein of Mycobacterium tuberculosis in pET SUMO plasmids as Vaccine Candidates
DOI:
10.29303/jppipa.v12i2.12724Published:
2026-02-28Downloads
Abstract
The Bacillus Calmette-Guerin vaccine did not show consistent results in preventing tuberculosis, with an effectiveness ranging from 0% to 80%. More effective proteins are needed as vaccine candidates to eliminate tuberculosis. Culture filtrate proteins 10-kDa(CFP-10) and Excretory Antigen Target 6-kDa(ESAT-6) demonstrated very strong antigenicity against T and B cells. These proteins are secreted during the early phase of infection and could potentially be used for vaccine candidates. This study aims to clone and express the CFP-10 and ESAT-6 proteins on the pET SUMO Plasmid. PCR with specific primers was used to amplify the genes encoding CFP-10 and ESAT-6 proteins. The PCR products were cloned into pET SUMO plasmids for transformation into competent cells E.coli BL21(DE3). Recombinant protein expression was induced in LB medium using 1 mM IPTG. The yield of recombinant proteins was visualized by SDS-PAGE and western blotting using an Anti-HisTaq Antibody. PCR results showed the target gene sizes to be 304 bp (CFP-10) and 291 bp (ESAT-6). SDS-PAGE and Western blotting revealed proteins at 22kDa (CFP-10) and 18kDa (ESAT-6). The genes encoding the CFP-10 and ESAT-6 proteins of M. tuberculosis have been successfully cloned into the pET SUMO plasmid and expressed in E. coli BL21(DE3) with molecular weights corresponding to the target genes.
Keywords:
CFP-10 ESAT-6 M. tuberculosis Vaccine CandidatesReferences
Abebe, F., Belay, M., Legesse, M., Mihret, A., & Franken, K. S. (2017). Association of ESAT-6/CFP-10-induced IFN-γ, TNF-α and IL-10 with clinical tuberculosis: evidence from cohorts of pulmonary tuberculosis patients, household contacts and community controls in an endemic setting. Clinical and experimental immunology, 189(2), 241-249. https://doi.org/10.1111/cei.12972.
Abdallah, A. M., Hill-Cawthorne, G. A., Otto, T. D., Coll, F., Guerra-Assunção, J. A., Gao, G., Naeem, R., Ansari, H., Malas, T. B., Adroub, S. A., Verboom, T., Ummels, R., Zhang, H., Panigrahi, A. K., McNerney, R., Brosch, R., Clark, T. G., Behr, M. A., Bitter, W., & Pain, A. (2015). Genomic expression catalogue of a global collection of BCG vaccine strains show evidence for highly diverged metabolic and cell-wall adaptations. Scientific reports, 5, 15443. https://doi.org/10.1038/srep15443
Anes, E., Pires, D., Mandal, M., & Azevedo-Pereira, J.M. (2023). ESAT-6 a Major Virulence Factor of Mycobacterium tuberculosis. Biomolecules, 13(6), 968. https://doi.org/10.3390/biom13060968
Bates, T. A., Trank-Greene, M., Nguyenla, X., Anastas, A., Gurmessa, S. K., Merutka, I. R., Dixon, S. D., Shumate, A., Groncki, A. R., Parson, M. A., Ingram, J. R., Barklis, E., Burke, J. E., Shinde, U., Ploegh, H. L., & Tafesse, F. G. (2024). ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages. bioRxiv : the preprint server for biology, 2023.08.16.553641. https://doi.org/10.1101/2023.08.16.553641
Chatterjee, S., Dwivedi, V. P., Singh, Y., Siddiqui, I., Sharma, P., Van Kaer, L., Chattopadhyay, D., & Das, G. (2011). Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner. PLoS pathogens, 7(11), e1002378. https://doi.org/10.1371/journal.ppat.1002378.
Ceylan, H., & Erdogan, O. (2017). Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag. Turkish Journal of Biology, 41: 77-87. https://doi.org/10.3906/by-1602-83
Fihiruddin., Artama, W. T., Wibawa, T & Mertaniasih, N.M. (2020). Cloning, expression and T cell epitope prediction of fbpA and fbpB genes of Mycobacterium tuberculosis clinical isolates. Journal of Microbiology Biotechnology and Food Sciences, 9(5),998-1002. https://doi.org/10.15414/jmbfs.2020.9.5.998-1002.
Forrellad, M. A., Klepp, L. I., Gioffré, A., Sabio y García, J., Morbidoni, H. R., de la Paz Santangelo, M., Cataldi, A. A., & Bigi, F. (2013). Virulence factors of the Mycobacterium tuberculosis complex. Virulence, 4(1), 3-66. https://doi.org/10.4161/viru.22329.
Gopal, G. J., & Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. The Protein Journal, 32:419-425. https://doi.org/10.1007/s10930-013-9502-5.
Guo, X., Du, W., Li, J., Dong, J., Shen, X., Su, C., Zhao, A., Wu, Y., & Xu, M. (2023). A Comparative Study on the Mechanism of Delayed-Type Hypersensitivity Mediated by the Recombinant Mycobacterium tuberculosis Fusion Protein ESAT6-CFP10 and Purified Protein Derivative. International journal of molecular sciences, 24(23), 16612. https://doi.org/10.3390/ijms242316612
Gyimah, F.T., & Dako-Gyeke, P. (2019). Perspectives on TB patients’ care and support: a qualitative study conducted in Accra Metropolis, Ghana. Globalization and Health. 15(1),19. https://doi.org/10.1186/s12992-019-0459-9.
Handayani, A.S., Astuti, T.W., Sartono, T.R., Arthamin, M.Z., & Tanoerahardjo, F.S. (2018). Immunogenicity Test of ESAT-6/CFP-10 Mycobacterium Tuberculosis (Indonesian Strain) Recombinant Protein Fusion: IFN-γ and CD8+ T Cells Expression in PBMC Culture. Journal Respirologi Indonesia, 38(4), 210-218
Indonesian Health Ministry. (2018). Profile of Indonesian Health 2018. Jakarta, Indonesia.
Jiang, Q., Zhang, J., Chen, X., Xia, M., Lu, Y., Qiu, W., Feng, G., Zhao, D., Li, Y., He, F., Peng, G., & Wang, Y. (2013). A novel recombinant DNA vaccine encoding Mycobacterium tuberculosis ESAT-6 and FL protects against Mycobacterium tuberculosis challenge in mice. Journal of biomedical research, 27(5), 406-420. https://doi.org/10.7555/JBR.27.20120114.
Jones-López, E. C., Namugga, O., Mumbowa, F., Ssebidandi, M., Mbabazi, O., Moine, S., Mboowa, G., Fox, M. P., Reilly, N., Ayakaka, I., Kim, S., Okwera, A., Joloba, M., & Fennelly, K. P. (2013). Cough aerosols of Mycobacterium tuberculosis predict new infection: a household contact study. American journal of respiratory and critical care medicine, 187(9), 1007–1015. https://doi.org/10.1164/rccm.201208-1422OC.
Kim, M. J., Ha, E. H., Lee, Y. K., Park, S. J., An, C. H., Park, S. Y., & Kim, S. K. (2022). ROP-ESAT6-CFP10 as Tuberculosis-Specific Antigen in Interferon Gamma Release Assay. Annals of clinical and laboratory science, 52(1), 126–132.
Koiri, D., Nandi, M., Abik, A.P.M., Aher, J.B., Kumar, A., Behura, A., Meher, G., Choudhary, V., Choubey, S., & Saleem, M. (2025). Real-time visualization reveals Mycobacterium tuberculosis ESAT-6 disrupts phagosome-like compartment via fibril-mediated vesiculation, Cell Reports, 44(3),
Kuo, C. J., Ptak, C. P., Hsieh, C. L., Akey, B. L., & Chang, Y. F. (2013). Elastin, a novel extracellular matrix protein adhering to mycobacterial antigen 85 complex. The Journal of biological chemistry, 288(6), 3886–3896. https://doi.org/10.1074/jbc.M112.415679.
Lu, W., Lin, J., He, Y., Yang, B., Qiu, F., & Dai, Z. (2025). Immunoregulation by ESAT-6: From Pathogenesis of Tuberculosis to Potential Anti-Inflammatory and Anti-Rejection Application. Pharmaceuticals (Basel, Switzerland), 18(9), 1408. https://doi.org/10.3390/ph18091408
Mahmoudi, S., Mamishi, S., Ghazi, M., Hosseinpour Sadeghi, R., & Pourakbari, B. (2013). Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens. Iranian journal of microbiology, 5(4), 374–378.
Malakar, B., Chauhan, K., Sanyal, P., Naz, S., Kalam, H., Vivek-Ananth, R. P., Singh, L. V., Samal, A., Kumar, D., & Nandicoori, V. K. (2023). Phosphorylation of CFP10 modulates Mycobacterium tuberculosis virulence. mBio, 14(5), e0123223. https://doi.org/10.1128/mbio.01232-23
Mertaniasih, N. M., Handijatno, D., Perwitasari, A. D. S., Dewi, D. N. S. S., Fanani, M. Z., & Afifah, I. K. (2016). Sequence analisys of the gene region encoding ESAT-6, Ag85B and Ag85C protein from clinical isolate of Mycobacterium tuberculosis. Procedia Chemistry, 18: 225-230. https://doi.org/10.1016/j.proche.2016.01.035.
Passos, B. B. S., Araújo-Pereira, M., Vinhaes, C. L., Amaral, E. P., & Andrade, B. B. (2024). The role of ESAT-6 in tuberculosis immunopathology. Frontiers in immunology, 15, 1383098. https://doi.org/10.3389/fimmu.2024.1383098
Peng, X., & Sun, J. (2016). Mechanism of ESAT-6 membrane interaction and its roles in pathogenesis of Mycobacterium tuberculosis. Toxicon : official journal of the International Society on Toxinology, 116, 29-34. https://doi.org/10.1016/j.toxicon.2015.10.003.
Piubelli, L., Campa, M., Temporini, C., Binda, E., Mangione, F., Amicosante, M., & Pollegioni, L. (2013). Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins. Microbial Cell Factories. 12:115. http://dx.doi.org/10.1186/1475-2859-12-115.
Purwanti, R., Indrati, A.R., Suraya, N., Montain, M.M., Atmosukarto, I., Nurainy, N., & Parwati, I. (2023). ESAT-6-Ag85C-polyHistag Antigen Fusion is Potential as Vaccine Candidate for Tuberculosis. The Indonesian Biomedical Journal, 15(2), 165-170
Ravimohan, S., Kornfeld, H., Weissman, D., & Bisson, G.P. (2018). Tuberculosis and lung damage; from epidemiology to pathophysiology. Eur Respir Rev. 27: 170077. https://doi.org/10.1183/16000617.0077-2017.
Seghatoleslam, A., Hemmati, M., Ebadat, S., Movahedi, B., & Mostafavi-Pour, Z. (2016). Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins. Iranian journal of medical sciences, 41(4), 296-304.
Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172. https://doi.org/10.3389/fmicb.2014.00172.
Ruhwald, M., de Thurah, L., Kuchaka, D., Zaher, M. R., Salman, A. M., Abdel-Ghaffar, A.-R., Shoukry, F. A., Michelsen, S. W., Soborg, B., Blauenfeldt, T., Mpagama, S., Hoff, S. T., Agger, E. M., Rosenkrands, I., Aagard, C., Kibiki, G., El-Sheikh, N., & Andersen, P. (2017). Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6. Scientific Reports, 7(45969). https://doi.org/10.1038/srep45969
Seifert, M., Vargas, E., Ruiz-Valdepeñas Montiel, V., Wang, J., Rodwell, T. C., & Catanzaro, A. (2021). Detection and quantification of Mycobacterium tuberculosis antigen CFP10 in serum and urine for the rapid diagnosis of active tuberculosis disease. Scientific reports, 11(1), 19193. https://doi.org/10.1038/s41598-021-98471-1
Shen, G. H., Chiou, C. S., Hu, S. T., Wu, K. M., & Chen, J. H. (2011). Rapid identification of the Mycobacterium tuberculosis complex by combining the ESAT-6/CFP-10 immunochromatographic assay and smear morphology. Journal of clinical microbiology, 49(3), 902–907. https://doi.org/10.1128/JMC.00592-10.
Solans, L., Aguiló, N., Samper, S., Pawlik, A., Frigui, W., Martín, C., Brosch, R., & Gonzalo-Asensio, J. (2014). A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component. Infection and immunity, 82(8), 3446–3456. https://doi.org/10.1128/IAI.01824-14.
World Health Organization. (2018). Global Tuberculosis Control: WHO Report 2018. World Health Organization Press, Geneva, Switzerland.
Wu, X., Wang, Y., Weng, T., Hu, C., Wang, F., Wu, Z., Yu, D., Lu, H., & Yao, H. (2017). Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis. Medicine, 96(51), e9350. https://doi.org/10.1097/MD.0000000000009350.
Xu, J. N., Chen, J. P., & Chen, D. L. (2012). Serodiagnosis efficacy and immunogenicity of the fusion protein of Mycobacterium tuberculosis composed of the 10-kilodalton culture filtrate protein, ESAT-6, and the extracellular domain fragment of PPE68. Clinical and vaccine immunology : CVI, 19(4), 536–544. https://doi.org/10.1128/CVI.05708-11.
Yuen, C. M., Weyenga, H. O., Kim, A. A., Malika, T., Muttai, H., Katana, A., Nganga, L., Cain, K. P., & De Cock, K. M. (2014). Comparison of trends in tuberculosis incidence among adults living with HIV and adults without HIV-Kenya, 1998-2012. PloS one, 9(6), e99880. https://doi.org/10.1371/journal.pone.0099880
Yindeeyoungyeon, W., Rukseree, K., Tungsudjai, S., & Panichakul, T. (2015). Evaluation of Recombinant Mycobacterium tuberculosis Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs. Advances in Microbiology, 5, 586-598. https://doi.org/ 10.4236/aim.2015.58061.
Zarif, R., Sankian, M., Gholubi, A., Farshadzadeh, Z., Soleimanpour, S., Youssefi, F., & Varasteh, A. R. (2013). Cloning and Expression of Mycobacterium tuberculosis Major Secreted Protein Antigen 85B (Ag85B) in Escherichia coli. Jundishapur Journal of Microbiology, 6(2),112-116. https://doi.org/10.5812/jjm.4701
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