Isolation and purification of recombinant bovine adenovirus type 3 hexone

Relevance. Adenoviruses (Mastadenovirus, Adenoviridae) are among the prevailing etiologic agents of the complex of respiratory and intestinal diseases of cattle, accounting for up to 70% of the nosological structure of morbidity in young animals. Diagnosis of adenovirus infection is difficult due to the diversity of serotypes, the most widely used in practice is enzyme immunoassay. It is possible to increase the sensitivity and specificity of the method by equipping test systems with purified recombinant antigens.

Methods. The work used methods of bioinformatics analysis and genetic engineering. Cloning of the synthesized insert encoding a fragment of the hexon gene was carried out in the pET-22b(+) vector, expression was carried out in E. coli organism. Classical methods of cell disintegration in combination with metal-chelate affinity chromatography were used to select purification conditions.

Results. Using classical methods of bioinformatics analysis of the nucleotide sequence of the WBR-1 strain of bovine adenovirus type 3, the epitope structure of the truncated recombinant hexon with a molecular weight of 17.0 kDa was developed. The protein with a hexahistidine tag was synthesized in a prokaryotic expression system by the producer strain E. coli BL21(DE3)pLysS/pET-22b(+)-Hexon in the form of inclusion bodies. Optimal conditions for culturing the strain and performing metal-chelate chromatographic purification on Ni-NTA Sepharose under denaturing conditions were selected, which made it possible to obtain an electrophoretically homogeneous product. Conditions for setting up an indirect enzyme immunoassay based on recombinant hexon were optimized. The methodological approach described in the work can be applied to obtain recombinant hexon as an antigen for obtaining polyclonal or monoclonal antibodies against adenovirus type 3, which is more effective and economical compared to the antigen of the native virus cultured in mammalian cells. 

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