glp-1 fusion protein comprising a mutated immunoglobulin fc portion
A technology of immunoglobulin and GLP-1, applied in peptide/protein components, medical preparations containing active ingredients, fusion polypeptides, etc., can solve the problems of short biological half-life and difficult concentration detection
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Embodiment 1
[0111] The construction of embodiment 1 carrier
[0112] Based on the above-mentioned technical route, adopt the method for molecular cloning, construct the following (from N-terminal to C-terminal) various expression vectors of the present invention:
[0113] GLP-1 analogue (amino acid sequence shown in SEQ ID NO.6)+peptide linker (amino acid sequence shown in SEQ ID NO.7)+mutated immunoglobulin Fc part.
[0114] The amino acid sequences of the constructed fusion proteins are respectively shown in SEQ ID NOs. 1-4, 8-11.
[0115] As a control, construct a vector encoding the following (from N-terminus to C-terminus):
[0116] For dulaglutide (WT), the amino acid sequence is shown in SEQ ID NO.5.
[0117] The mutated immunoglobulin Fc portion of the present invention is summarized as follows:
[0118] Table 1
[0119]
[0120] The nucleotide sequences encoding the WT fusion protein (dulaglutide) and the IgG4-Fc mutant fusion protein were obtained by chemical synthesis ba...
Embodiment 2
[0121] Example 2 Vector transfection and expression in cells
[0122] After recovering CHO host cells with CHO medium, when the cell density is about 8x10 5 Cells were harvested for transfection at 1 cells / mL. Transfected cells about 1x10 7 Each cell was transfected with about 40 μg of plasmid (Bio-Rad, Genepulser Xcell) by electroporation. Cells were cultured in 20 mL CHO medium after electric shock. On the second day of culture, the cells were collected by centrifugation, and resuspended in 20 mL of CHO medium added with G418 (Geneticin) to a final concentration of 800 μg / mL. When the cell density is about 0.6x10 6 cells / mL, the obtained mixed clones were subcultured in CHO medium, and the subcultured cell density was about 0.2x10 6 cells / mL. When the cell survival rate was about 90%, the cell culture fluid was collected.
Embodiment 3
[0123] Embodiment 3 purifies fusion protein from animal cell culture fluid
[0124] A series of fusion proteins in Example 1 were tested at the translation level. Use Protein A filler to enrich a small amount of cell culture fluid, and collect the fusion protein. The obtained fusion protein is a homodimer formed by disulfide bonds and various non-covalent interactions. Mass spectrometry is used to detect the molecular weight. About 62KD, consistent with the theoretical molecular weight. The collected fusion protein was purified using Protein A chromatography column. The collected samples were detected by 10% SDS-PAGE electrophoresis after reduction, and the electrophoresis pattern showed a single band, about 36KD. Purified samples were dialyzed overnight at 4°C against 10 mM PBS buffer, pH 7.2.
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