Development of DNA-Designed Avian IgY Antibodies for Detection of Mycobacterium avium subsp. paratuberculosis Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Cattle

• Published in: Applied biochemistry and biotechnology 2012-05, Vol.167 (1), p.14-23
• Main Authors: Nikbakht Brujeni, Gholamreza, Gharibi, Darioush
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Bacterial - genetics; Antibodies, Bacterial - immunology; Antibody Specificity; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Biochemistry; Biological and medical sciences; Biotechnology; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - immunology; Cattle Diseases - microbiology; Chemistry; Chemistry and Materials Science; Chickens; Deoxyribonucleic acid; DNA; Female; Fundamental and applied biological sciences. Psychology; Heat shock proteins; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - immunology; Immunization; Immunoglobulins - genetics; Immunoglobulins - immunology; Male; Monoclonal antibodies; Mycobacterium avium; Mycobacterium avium subsp. paratuberculosis - genetics; Mycobacterium avium subsp. paratuberculosis - immunology; Mycobacterium avium subsp. paratuberculosis - isolation & purification; Paratuberculosis - diagnosis; Paratuberculosis - immunology; Paratuberculosis - microbiology; Poultry; Vaccines
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-012-9648-1

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 ( MAP Hsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAP Hsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1- MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1- MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAP Hsp70, and in a sandwich ELISA to assess the prevalence of anti- MAP Hsp70 antibodies in cattle serum. Western blotting results indicate the expression of r MAP hsp70 in COS-7 cells and sandwich ELISA could detect anti- MAP Hsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1- MAP Hsp70 could demonstrate the effective production of anti- MAP Hsp70 IgY antibodies. Monospecific anti- MAP Hsp70 antibody generated in chickens is useful for detection of MAP Hsp70 peptide in cell culture and MAP lysate.