Cloning of Mycobacterium avium paratuberculosis hsp70 gene into pET-24a plasmid and its expression in Escherichia coli

Subject Areas : Veterinary Clinical Pathology

Rasa Sheini mehrabzade ¹ (Ph.D. Student in Bacteriology, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.)
Masoudreza Siefi abad shapori ² (Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.)
Masoud Ghorbanpoor ³ (Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.)
Darush Gharibi ⁴ (Associate Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.)

Received: 2022-04-29 Accepted : 2022-08-13 Published : 2022-11-22

Keywords: cloning, Johne's disease, Heat Shock Protein 70, Mycobacterium avium paratuberculosis, pET-24a plasmid,

Abstract :

Johne's disease (Paratuberculosis) is a disease caused by Mycobacterium avium paratuberculosis (MAP) which is a chronic and progressive infection that chiefly affects ruminants. The disease can lead to significant economic losses in the livestock industry and may also be a threat for public health; because it may be transmitted to humans through consumption of milk and other contaminated animal products. Immunity and resistance against the Johne's disease is mainly due to cell mediated immune response. Heat Shock Protein 70 (HSP70) of this bacterium is one of its important proteins, which the immune response against it can prevent the fecal excretion of bacteria. In order to facilitate the production of recombinant vaccine against Johne's disease, in this study the recombinant HSP (recombinant HSP; rHSP) was produced and its immunogenicity investigated in rabbits. For this purpose, the hsp70 gene was cloned into pET-24a plasmid and the resulting recombinant plasmid was transferred to E. coli strain BL21. The expression of the above protein was checked by SDS-PAGE and the accuracy of the nucleotide sequence was confirmed by sequencing. Immunization of rabbits by rHSP70 resulted in the production of high levels of antibodies. Based on the findings, it seems that the HSP70 specific antibody can be evaluated in the design of diagnostic methods of the disease and the produced recombinant protein can be assessed for the production of recombinant vaccines.

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