Preparation and determination of the serological activity of recombinant brucella O-antigen synthesized in the eukaryotic expression system

Brucellosis is a dangerous zoonotic disease caused by bacteria of the genus Brucella. having a significant negative impact on the productivity of farm animals. In the Russian Federation, successes in the fight against this disease in cattle are associated with the use of a vaccine based on the Brucella abortus 82 strain, as well as the timely detection of infected animals. This work is devoted to the preparation and study of the serological activity of recombinant O-antigen for the indication of antibodies against brucellosis. As a result of conducting search queries in the GenBank database, we identified the amino acid sequence of the O-polysaccharide antigen brucella –WP_002967174.1. This sequence was optimized for protein translation in mammalian cell culture, synthesized, and cloned into the pVax1 plasmid. pVax1 was developed by transformation of Escherichia coli cells of the DH5a strain by heat shock, with selection for the kanamycin resistance gene with further isolation and purification of plasmid DNA. Transfection of HEK293 cells with the resulting genetic construct led to the expression of the target antigen, the presence of which in the liquid taken from the cell culture was established using vertical electrophoresis in 12% PAAG based on the visualization of major patterns at the level of 70 kDa. As a result of ion exchange and gel filtration chromatography, it was possible to obtain purified O-antigen in dissolved form at a concentration of 4.7 mg/ml. The resulting drug was used in a dilution of 10 micrograms/ml, as an antigen, during indirect solid-phase ELISA to indicate antibodies in bovine blood sera against S and R brucella antigen. The coefficient of specificity was calculated with respect to ELISA data with blood serum at a dilution of 1:256. Thus, in the reaction with bovine blood serum against Brucella abortus, the specificity coefficient was 6.29, against S and R brucella antigens – 6.1 and 6.77, respectively. The results of the study can make a significant contribution to the development of methods for the diagnosis and treatment of brucellosis, as well as to the creation of new vaccine preparations.

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