OBTAINING AND DETERMINATION OF IMMUNOGENIC PROPERTIES OF TRX-PD-1 RECOMBINANT PROTEIN
Main Article Content
Authors
Zh. Adish
National Center for Biotechnology, 13/5, Korgalzhyn road, Nur-Sultan, 010000, Kazakhstan
K.N. Mukantaev
National Center for Biotechnology, 13/5, Korgalzhyn road, Nur-Sultan, 010000, Kazakhstan
K.A. Tursunov
National Center for Biotechnology, 13/5, Korgalzhyn road, Nur-Sultan, 010000, Kazakhstan
D.B. Kanaev
National Center for Biotechnology, 13/5, Korgalzhyn road, Nur-Sultan, 010000, Kazakhstan
Ye.M. Ramankulov
National Center for Biotechnology, 13/5, Korgalzhyn road, Nur-Sultan, 010000, Kazakhstan
K.K. Mukanov
National Center for Biotechnology, 13/5, Korgalzhyn road, Nur-Sultan, 010000, Kazakhstan
Abstract
Monoclonal antibodies against programmed cell death receptor PD-1 are of particular interest for immunotherapy and blocking control points for tumor development. The PD-1 T-cell receptor is a regulator capable of inhibiting or completely suppressing the immune response. One of the important stages of obtaining monoclonal antibody is the immunization of BALB/c mice, the scheme of which depends on the nature of the antigen and its immunogenicity.
The gene of PD-1 was synthesized by a two-step polymerase chain reaction using the Phusion High-Fidelity DNA Polymerase. The resulting construction based on the pET32 vector was transformed by electroporation into the E. coli BL21 expression strain, which resulted in the E. coli strain BL21/pET32/Trx-PD-1. Molecular weight of the protein Trx-PD-1 was 34 kDa. Western blot demonstrated presence of a hexahistidine tag in a protein with a molecular mass of 34 kDa. The highest antibody titers were observed in mice immunized with recombinant protein at a concentration of 100 μg/ml. Western blot revealed a specific reaction of PD-1 protein without thioredoxin with sera from immunized mice.
The resulting construct pET32/PD-1 provided a high level of expression of the recombinant Trx-PD-1. The recombinant Trx-PD-1 induced high titers of antibody and stimulated B-lymphocytes.
Keywords
PD-1, extracellular domain, prokaryotic expression, recombinant protein, antibodies
Article Details
References
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