Development of a Test System Based on Recombinant GM6 Antigen from Trypanosoma evansi for the Determination of Surra in Horses
Development of a Test System Based on Recombinant GM6 Antigen from Trypanosoma evansi for the Determination of Surra in Horses
Nurlan Akhmetsadykov1*, Tanatar Kydyrov2, Moldir Akhmetzhanova1, Gulnazi Akhmetova3, Maxat Berdikulov4
ABSTRACT
Trypanosomosis, such as Surra, causes significant damage to livestock worldwide. The development of new serological diagnostic methods is highly relevant. The primary objective of this study was to produce a recombinant GM6 antigen derived from T. evansi and investigate its immunological properties for the detection and diagnosis of trypanosomosis. To create a recombinant antigen, bioinformatic analysis of the primary structure of T. evansi GM6 antigen from several regions was carried out. The protein sequence was reverse translated to the nucleotide sequence, after which the gene was synthesized using solid-phase method. The gene fragment, encoding the GM6 protein, was inserted or cloned into the pET28 expression vector after synthesis. The obtained sequences were checked by sequencing for correspondence to the matrix molecule. For transformation E. coli BL21 (DE3) strain was used. To assess the efficacy of the antigen, immunoassay analysis and blot-hybridization techniques were employed. Obtained results showed that 1mM isopropyl ß-D-1-thiogalactopyranoside induction at 2℃ for 18 hours was the most optimal condition for expression of recombinant GM6 T. evansi antigen. From a 500 ml of growth medium, a total of 3.4 mg of recombinant protein was successfully obtained. A recombinant GM6 antigen of T. evansi encoding a 30 kDa polypeptide was developed, which has diagnostically significant sensitivity in the enzyme immunoassay at a dilution of 1:6400 with sera of horses and donkeys infected with trypanosomosis. The proposed test system can be used for diagnosis of trypanosomosis in the early stages of the disease.
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