关键词: TIGIT, 融合蛋白, 真核表达, 自然杀伤细胞

Abstract: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is a novel immunosuppressive receptor, which plays an important role in the immune regulatory network of the body. In order to further explore the immunomodulatory function of TIGIT in natural killer (NK) cells, a eukaryotic expression vector for fusion protein composed by TIGIT extracellular domain (TIGIT amino acids 1~135, to retain the functional domain IgV region, this extracellular segment is simply called TIG) and Fc segment of human immunoglobulin G3 (IgG3) was constructed, and the expression of TIG-Fc fusion protein and its effect on NK-92 cell function were preliminarily studied. Using molecular biology method, the gene carrying human TIG and Fc segment of IgG3 were fused, and then inserted into eukaryotic expression vector pcDNA3.1(-) to construct recombinant plasmid pcDNA3.1(-)-TIG-Fc. Subsequently, the recombinant plasmid was transfected into human embryonic kidney (HEK) 293T cells, and the expression of TIG-Fc fusion protein in 293T cells was detected by flow cytometry and Western blot. Then the recombinant plasmid was transfected into NK-92 cells, and the effect of TIG-Fc fusion protein on NK-92 cell proliferation was detected by WST-1, and the effect of TIG-Fc fusion protein on IFN-γ secretion level of NK-92 cells was detected by ELISA. The results showed that the eukaryotic expression vector for human TIG and human IgG3 Fc fusion expression was successfully constructed, and the expression of TIG-Fc fusion protein could significantly inhibit the proliferation of NK-92 cells and their secretion level of IFN-γ (P<0.05), which laid an important foundation for the functional research of TIGIT in the later stage.

Key words: TIGIT, fusion protein, eukaryotic expression, NK cell