The theoretical and practical aspects of the recombinant antigens for the diagnosis of bovine leukemia production

Abstract

Introduction. Nowadays the problem of bovine leukemia (EBL) effective diagnosis in countries where EBL is registered and the disease-free areas remains actual. The main diagnostic tests are immunodiffusion reaction (AGID) and enzyme-linked immunosorbent assay (ELISA), which allow the identification of infected animals by the presence of antibodies to bovine leukemia virus (BLV)  both in serum and in milk samples. The effectiveness of these methods depends on the quality of diagnostic test systems used and determines by the cultural and recombinant virus antigens specificity. EBL recombinant antigens have certain advantages as they are more active and cheap. Purpose of the work. The analysis of theoretical and practical approaches in the bovine leukemia virus recombinant antigens development and its diagnostic potential evaluation. The article contains data from the literature on the recombinant antigens of bovine leukemia virus construction and use. Analysis of the literature showed that the recombinant proteins are widely used in the serological diagnosis of bovine leukemia. Numerous protocols of BLV gp51 and p24 immunodominant antigens preparation has been  developed in heterologous systems (Saccharomyces cerevisiae, E. coli, vaccinia virus, baculovirus). In order to obtain recombinant antigens, the BLV provirus genome regions isolated from FLK-BLV cell culture, lymphocytes or tumor cells from naturally infected cattle are typically used. For the recombinant antigens labeled by hexahistidine or Srept II purification one-step immobilized-metal affinity chromatography IMAC and highly selective Strep-Tactin affinity chromatography methods are carried out. The end products activity and specificity are studied in the immunoblotting, ELISA and AGID diagnostic reactions.  The ukrainian scientists’ publications are devoted to the  clone E. coli HB101-2 transformed by the recombinant plasmid containing fully functional BLV env and gag genes nucleotide sequence construction. The efficiency of BLV gp51 and p24 encoding regions fusion-sequence integration was confirmed by  the screening with the specially designed oligonucleotides. The recombinant antigen expression was induced by addition of IPTG. To isolate the antigen bacterial mass was destroyed by defrostation and ultrasonic disintegration in the experimentally selected modes. The activity and specificity of the antigen was determined by AGID with the use of the bovine fetal serum, positive and negative  reference diagnostic serum by  unified method in comparison with the standardized cultural BLV antigen in AGID. The antigen  specificity was increased by adsorption with commercial anticolibacillosis serum. The antigen activity was confirmed by AGID. Conclusions. Nowadays the most promising BLV antigens expressing genetic constructions with E. coli and baculovirus. E. coli recombinant strains are  the most available and effective using expressing system, which allows to get an active and specific antigenic product if  an optimal vector constructions and commercially available systems of metal affinity chromatography purification and control  with appropriate Mab are used. As an cultural  and recombinant antigens alternative the mimicking critical BLV antigenic epitopes synthetic peptides were tested. In recent times  many scientific works formed the basis for the bovine leukemia diagnostic test systems’ creation, which are now widely available on the biotechnological products market. Although the majority of manufacturers prefer the recombinant antigens of the pathogen, pilot studies on more improved and cheaper ways to obtain different diagnostic antigens preparations shall not lose relevance.