Abstract
BackgroundMycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease in all ruminants worldwide. Economic problems in dairy cattle and sheep industries, public health concern, persistence of MAP in the environment and lack of effective vaccines mentioned necessity of research about various antigens to introduce as vaccine candidates. Based on MAP pathogenesis, it seems that research about the production of new recombinant proteins to stimulate cell-mediated immunity is helpful. This study describes successful expression and purification of a chimeric fusion protein which consists of Heparin-Binding Hemagglutinin Adhesin (HBHA) and high antigenic region of Fibronectin Attachment Protein (FAP-P). Triggered antigen-specific IFN-γ response of isolated PBMCs from immunized goats to rHBHA-FAP and all crude proteins of MAP (PPD), was measured by ELISA.ResultsSignificant increases were observed in the IFN-γ production level of peripheral blood mononuclear cells (PBMCs) stimulated by constructed chimeric protein from rHBHA-FAP and PPD vaccinated goats. Antigen-specific gamma interferon (IFN-γ) secretion in positive group (immunized by PPD) against rHBHA-FAP and test group (immunized by rHBHA-FAP) against PPD, also statistically insignificant rises between stimulation with rHBHA-FAP and PPD, suggested the potential and specificity of our chimeric protein to stimulate cell mediated immunity against MAP.ConclusionsCollectively, these results demonstrate that rHBHA-FAP elicits a strong IFN-γ production in PBMC culture. Therefore, further studies of the present product as a candidate vaccine in naturally infected animals should be conducted, to analyze its potential.
Highlights
Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease in all ruminants worldwide
Plasmid construction and cloning The designed chimeric gene consists of the Heparin-Binding Hemagglutinin Adhesin (HBHA)-coding sequence, (Pro Glu)7 as a linker, and high antigenic region of fibronectin attachment proteins (FAPs)-P was synthesized and inserted in pUC57 between restriction sites of EcoRI and HindIII by GenScript company (USA)
Successful expression and purification of rHBHA‐FAP in E. coli BL21 (DE3) The fusion gene was successfully subcloned into the pET26b, transformed into E. coli BL21 (DE3), and confirmed by colony PCR
Summary
Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease in all ruminants worldwide. There are studies about the association of MAP with Crohn’s disease [7], sarcoidosis and Blau syndrome [8], type 1 diabetes [9], Hashimoto’s thyroiditis [10], and multiple sclerosis (MS) [11] This could explain the significant risk of MAP to public health safety. HBHA is located on the surface of mycobacteria and is important in the binding of mycobacteria to the epithelial cells [19] during bovine tuberculosis and Johne’s disease; it is a major target for host humoral immune response. The FN-binding glycoprotein family including fibronectin attachment proteins (FAPs) is important for attachment and internalization of MAP by epithelial cells and induce T h1 polarization and IFN-γ production in vitro [24]
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