Cloning, expression and purification of ESAT-6 protein from Mycobacterium bovis strain AN5 and evaluation of immunogenicity of two recombinant proteins ESAT-6 and CFP-10 in guinea pigs.

Zahraei Salehi, Taghi; Arefpajoohi, Reza; Mosavari, Nader; Salehi Najafabadi, Zahra; Yahyaraeyat, Ramak

doi:10.22034/nfvm.2026.560371.1303

Cloning, expression and purification of ESAT-6 protein from Mycobacterium bovis strain AN5 and evaluation of immunogenicity of two recombinant proteins ESAT-6 and CFP-10 in guinea pigs.

Document Type : Original Article

Authors

Taghi Zahraei Salehi ¹

Reza Arefpajoohi ²

Nader Mosavari ³

Zahra Salehi Najafabadi ⁴

Ramak Yahyaraeyat ²

¹ Department of Microbiology and Immunology, Faculty of veterinary medicine, University of Tehran, Tehran, Iran.

² Department of Microbiology and Immunology, Faculty of Veterinary Medicine,

³ Department of Research and Production of Tuberculin and Mallein, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization, Karaj, Iran

⁴ Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization, Karaj, Iran

10.22034/nfvm.2026.560371.1303

Abstract

The basis for diagnosing cattle tuberculosis is the skin test or tuberculin test. However, the presence of false positive cases in this skin test due to the occurrence of cross-reactions of PPD prepared from the AN5 strain of Mycobacterium bovis with other mycobacteria is considered one of its disadvantages, which is why the use of proteins with higher specificity is recommended to overcome it. One of the objectives of this study was to clone, express, and purify the recombinant ESAT-6 protein and evaluate its immunogenicity as an important antigen of Mycobacterium bovis in the prokaryotic system. In this study, the esat-6 gene encoding the secretory protein ESAT-6 was amplified using the polymerase chain reaction method. Then, the obtained PCR product was digested with two restriction endonuclease enzymes EcoRI and HindIII and then cloned in the plasmid vector pET23a(+) and amplified after transformation in Escherichia coli DH5α. In order to express the recombinant ESAT-6 protein, the above vector containing the cloned esat-6 gene was transferred to Escherichia coli expressing BL21. The correctness of the gene cloning was confirmed by sequencing. In the following study, the recombinant ESAT-6 protein was purified using nickel resin and its antigenic expression was confirmed by Western blotting using specific monoclonal antibodies. The results of gene sequencing along with Western blotting indicated that the obtained recombinant protein was well expressed and purified and could be used in challenge experiments and diagnostic tests like the recombinant CFP-10 protein.

Keywords

ESAT-6 protein

CFP-10 protein

Mycobacterium bovis

Mycobacterium tuberculosis complex

Subjects