Variable domain of the heavy chain of heavy-chain antibody of the Rv0733 antigen of mycobacterium tuberculosis panned and identified from a nonimmune llama VHH phage display library

The ability of Mycobacterium tuberculosis to cause disease is closely correlated with its ability to enter into and survive inside, what is currently thought to be, it's permitted host cell, the macrophage. Rv0733 is one of the key factors for living M. tuberculosis to maintain intracellular nucleotide pools. Variable domain of the heavy chain of heavy-chain antibody (VHH) is one of the essential molecules for neutralizing antigens of the intracellular pathogen. Recombinant nonimmune Llama VHH phage display antibody library was enriched after three rounds of panning with M. tuberculosis Rv0733-6His fusion antigen. The size of the VHH phage library was reduced from 1.5x10(9) to 4x10(3), but the antigen-binding activity increased from 0.3-0.6 to 2.5-2.8. Eleven independent sequences were obtained from 1024 enriched clones by phage enzyme-linked immunosorbent assay and sequencing technology. The recombinant expression vector constructed by cloning Rv0733-VHH gene into the prokaryotic expression plasmid pET-22b-IFH harboring Fc portion of human IgG and 6His-tag gene was transformed into Escherichia coli for induction and expression of fusion antibodies to determine their potential in binding with Rv0733 antigen. The purity and the size of the expressed Rv0733-VHH-Fc-6His antibodys were confirmed by the Western blot. Immunofluorescence staining confirmed the specificity of the Rv0733-VHH-Fc-6His antibody binding to Rv0733 antigen. Treatment of macrophages with pcDNA3.1-Rv0733-VHH -6His led to reduced mycbacterial loads 0.31-fold (P < 0.05) as compared with control cells by day 7 post-infection. These findings may provide an alternative approach for further studies on Rv0733-VHH antibody binding with intracellular M. tuberculosis antigen and improve the bactericidal ability of macrophages.