Abstract
Effective vaccines for infectious animal diseases, including zoonotic animal diseases of livestock and poultry, including brucellosis of ruminants, are often not available or not efficacious. This work presents a targeted approach and methodology to identify potential protein and peptide vaccine candidates. This methodology applies to all microbial organisms for which procedures are developed to study membrane proteins in particular. Certain microbial membrane proteins are important receptors of signal proteins of the host’s immune response system for the initiation of a protective immune response or alternatively, some membrane proteins are activated to allow the organism to escape the host’s immune surveillance system. Here we present an approach to directly target the membrane proteins which might afford effective protection. A membrane protein extraction procedure based on previously published procedures by investigators separates three fraction of membrane proteins using TX-100 differential protein extraction procedures. This method results in three types of protein fractions, detergent-soluble proteins, aqueous soluble proteins (including periplasmic proteins), and detergent-insoluble proteins. One and two-dimensional electrophoresis separates (and purifies) these proteins. Western Blots with antibodies from infected and naïve animals as controls are compared and appropriate protein bands or spots are excised, digested with a variety of proteolytic enzymes, and submitted for MALDI-TOF spectrometry. Analysis of the data with SwissProt database programs provides the protein ID and sequence. The identified sequences are then further analyzed by three-dimensional modeling using MolBio and protein external protein loops are identified. Sequences are further studied for loop/sequence homologies and function by the publicly available Resource Center of the Zhang Laboratory which identifies additional potential functionalities of the identified protein(s), providing a better understanding of the immune response regulation. Examples of proteins and peptides identified by this approach are presented.
