Immunomodulatory fusion proteins
A technology of fusion protein and immunoglobulin, applied in the fields of cytokine receptor fusion protein, immunology and fusion protein, which can solve problems such as existing demand
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Embodiment 1
[0110] Embodiment 1: IL-10RA fusion protein particle construction
[0111] This example describes the generation of plasmid and viral expression vectors encoding IL-10RA fusion proteins.
[0112] Nucleotide sequences encoding a series of human IL-10RA fusion proteins were generated. The first fusion protein hIL-10R-IgG1 (SEQ ID NO: 58) includes nucleotide residues 1-229 of human IL-10RA (ending at SLTRQ), followed by nucleotide residues 84-330 of the human IgG1 sequence. residues (starting with NVNHK). The second fusion protein hIL-10R-Fc (SEQ ID NO: 48) includes residues 1-235 of human IL-10RA (ending at FTVTN), followed by residues 104-324 of human IgG1 (ending at at DKTHT). Details of the fusion proteins are shown in Table 3.
[0113] table 3
[0114]
[0115] The nucleotide sequence encoding the fusion protein is optionally cloned into a plasmid for downstream applications. Specifically, recombinant adenoviral vectors expressing hIL-10R-IgG1 or hIL-10RA-Fc without...
Embodiment 2
[0116] Embodiment 2: IL-10R fusion protein activity
[0117] A549 cells (human lung cancer cells) were infected with the virus vectors described in Example 1 not expressing the transgene but expressing hIL-10R-IgG1 or hIL-10RA-Fc at 10 MOI and cultured for four days. Collect the modulated medium from the cell culture, and THP-1 cells (human leukemia mononuclear cells) are divided into 5×10 6 cells / ml were suspended in the conditioned medium. Cells were treated with 50 ng / ml human IL-10 for 30 minutes at 37°C, or left as is as a control. To measure IL-10 activity, extracted intracellular proteins from THP-1 cells were probed for phosphorylated Stat3 by Western blot. Total Stat3 was used as an internal reference.
[0118] IL-10 induced Stat3 phosphorylation in THP-1 cells cultured in conditioned medium from cells infected with a viral vector not expressing the transgene or expressing hIL-10RA-Fc. However, IL-10 did not induce Stat3 phosphorylation in THP-1 cells cultured in ...
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