Development and molecular analyses of Xanthomonas pthA specific scFv recombinant monoclonal antibodies

Abstract

The Xanthomonas citri pv. citri (Xcc) is causal agent of bacterial citrus canker which is major disease of citrus throughout the world. The pthA bacterial effector protein is presented within the infected plants and indispensable of canker. The scFv antibodies are valuable tools for diagnosis and suppression of pathogens within plants. The present article describes developing and characterization of specific recombinant monoclonal scFv antibodies against pthA effector protein. For this aim, the gene encoding pthA protein was heterologously expressed in Escherichia coli and used for screening of Tomlinson phage display antibody library to pinpoint specific single chain variable fragment (scFv). In each round of panning, the affinity of phage towards pthA was checked by enzyme linked immunosorbent assay (ELISA). The data was indicative of about 50% of the monoclonal phages to be reactive strongly against pthA protein. Among the positive clones, 5 samples (A12, B8, C1, H8 and G8) were capable of detecting Xcc-infected plant samples and recombinant pthA protein. Restriction fragment length polymorphism showed similar banding pattern for all 5 scFvs as renamed to pthA-scFG8. HB2151 E. coli cells were infected by the phage bearing pthA-scFG8, and the expression of the peptide was induced by IPTG to produce a 30 kDa recombinant molecule. I-TASSER was used for homology modeling of both scFv and pthA and docking was carried out by Hex program. The latter demonstrated binding energy of −784 kcal/mol in scFv-pthA.

Keywords

• biopanning
• citrus bacterial canker
• phage display
• single chain fragment variable