Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis

Document Type: Original article

Authors

1 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University, Chaumuhan, Mathura, UP, India 281406

Abstract

The aim of this study is to identify, clone and express a Mycobacterium avium subsp. paratuberculosis specific immunogenic antigen candidate, in order to develop better reagents for diagnosis and vaccines for the protection of the host. Therefore, MAP2191 gene (a member of MAPmce5 operon) from MAP, was isolated and characterized by Bioinformatics tools and in vitro experiments. Then, a novel Mce-whole protein encoded by MAP2191 gene was amplified and sub-cloned into E. coli. We tried to express the Mce/whole protein in different condition along with a positive expression control (pET28a-Mce/truncated plasmid that we know express well), to ensure that nothing is wrong regarding culture/induction condition. The level of the recombinant protein expression was analyzed by means of SDS-PAGE and Western blotting. Western blot analysis toward full-length MAP2191 protein and its truncation only demonstrated Mce/truncated protein. The concurrence of in-silico prediction of primary structure of MAP2191 protein results along with experimental results confirmed that expression of Mce/whole protein was affected by the hydrophobicity nature of this protein. Our data support the hypothesis that the presence of hydrophobic regions in protein structure can influence the level of recombinant protein expression. This stresses the importance of gene selection and the protein sequence checking of the hydrophobic content in any protein purification project in order to achieve a large amount of desirable proteins.

Keywords


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