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.

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The GPR161 is a receptor of the GPCR family and identified as a prognostic biomarker for triple negative breast cancer (TNBC). TNBC characterized by lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor (Her2), early recurrence and poor prognosis. The GPR161 is an important regulator of the proliferation and migration of cancer cells. However, anti-GPCR receptor drugs are rarely used in the treatment of cancer, despite evidence of receptor involvement in various aspects of cancer development. Upon receipt of monoclonal antibodies, the success of the study is associated with the presence in sufficient quantities of pure antigen preparations. Recently, gene synthesis under de novo conditions has become a powerful tool in biotechnology. A number of methods have been described, representing thermodynamic balanced methods based on polymerase chain reaction (PCR). This paper presents the results of gene synthesis and the preparation of a recombinant extracellular fragment of the receptor. To obtain a recombinant protein, an area located outside the cell membrane between the 4th and 5th helix of the receptor was selected. A genetic construct based on the pET32 vector carrying the extracellular fragment of the human GPR161 receptor has been developed and obtained. Strain Escherichia coli BL21/pET32/TM4-5GPR161 producing recombinant extracellular fragment rTM4-5 GPR161 was obtained. The conditions for the isolation and purification of recombinant protein were determined, and its immunogenic properties were studied. Purified recombinant protein TM4-5GPR161 in its immunological characteristics suitable for the production of strains of hybrid cells producing monoclonal antibodies to the GPR161 receptor.