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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #333464

Research Project: INVASIVE PATHOGENS OF CITRUS

Location: Molecular Plant Pathology Laboratory

Title: Characterization and purification of proteins suitable for the production of antibodies against ‘Ca. Liberibacter asiaticus’

Author
item Liu, Huawei - China Agriculture University
item Atta, Sagheer - University Of The Punjab
item Hartung, John

Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2017
Publication Date: 7/18/2017
Citation: Liu, H., Atta, S., Hartung, J.S. 2017. Characterization and purification of proteins suitable for the production of antibodies against ‘Ca. Liberibacter asiaticus’. Protein Expression and Purification. 139:36-42.

Interpretive Summary: Citrus greening is an exceptionally dangerous disease of orange, grapefruit and lime trees. Antibodies are exceptionally useful tools for the detection of plant pathogens, but until recently have not been available for the bacterium that causes citrus greening disease. This is because the bacterium has not been grown in the laboratory. We decided to make antibodies against this bacterium, and as a first step in this work we used computer data to identify six proteins that are present on the surface of the bacterium. We also put the genes that make these proteins into another bacterium that can be grown in the laboratory. Finally we used a trial and error approach to identify the best ways to purify the proteins so that they could be used to make antibodies. These proteins have been used to make antibodies by injecting rabbits by standard methods. The antibodies that were made possible by this work have been developed into useful diagnostic tests for the pathogen. Our results will be used by scientists that carry out research on citrus greening disease.

Technical Abstract: The citrus disease huanglongbing (HLB), which is caused by ‘Candidatus Liberibacter asiaticus’ (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several 'CaLas' proteins that provide the basis for efficient and accurate detection of 'CaLas' in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies. The current report details purification procedures for six protein antigens used to select recombinant and polyclonal antibodies. These proteins include a flagellar biosynthesis protein (FlhA), a dinucleoside polyphosphate hydrolase (InvA), a portion of the major outer membrane protein (OmpA), a component of type IV pilus (CapB), the polysialic acid capsule expression protein (KpsA) and the outer membrane efflux protein (TolC). Different purification conditions were required for each of the different proteins. Bioinformatic analysis indicated there were great differences in the secondary and three-dimensional crystal structures of the six antigens, which likely contributed to the need for different purification conditions. The results of the bioinformatic analysis also indicated that the six proteins contained a great diversity of antigenic epitopes, which varied in number, and that the antigenic clusters were not uniformly distributed throughout the proteins. Such data could be extremely useful for the development of highly specific antibodies capable of differentiating specific strains of Liberibacter.