Glucagon like peptide-1 mutant polypeptide and preparation method, medicinal composition and use thereof
A glucagon, mutant technology, applied in the direction of glucagon, drug combination, hormone peptide, etc., to achieve the effect of improving compliance attitude
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[0052] Another aspect of the present invention also provides a method for preparing the above-mentioned GLP-1 mutant polypeptide, the method comprising: after the GLP-1 mutant polypeptide is prepared in solid phase by a polypeptide synthesizer, adding 50-100 mM DTT (dithiothreitol) or β-Mercaptoethanol is denatured, and then slowly oxidized at 4°C or fed with oxygen to form a disulfide bond.
[0053] Another aspect of the present invention also provides the application of the above-mentioned GLP-1 mutant polypeptide in the preparation of a drug for treating diabetes, and the application of the polypeptide complex in the preparation of a drug for treating and / or preventing obesity.
[0054] Moreover, the pharmaceutical composition is preferably an injection, more preferably a lyophilized powder injection or a solution injection.
[0055] Another aspect of the present invention also provides a pharmaceutical composition comprising the above-mentioned GLP-1 mutant polypeptide.
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Embodiment 1
[0084] Example 1 In vitro GLP-1 receptor binding experiment of GLP-1 mutant polypeptide
[0085] The receptor binding experiment was carried out using the GLP-1 receptor high expression cell line INS-1 (rat islet cell-1, purchased from the Animal Center of Shanghai Chinese Academy of Sciences). First, INS-1 cells were seeded on a 12-well plate, and the cells were washed with PBS buffer 2 hours before the experiment, and then the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After cell counting, 10 5 Cells / hole were respectively in GLP-1 mutant polypeptide (SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8 and SEQ ID9) and natural GLP-1 (SEQ ID ID NO 1) Co-incubate for 2 hours. After washing the unbound polypeptides with PBS, the GLP-1 ELISA kit was used for detection. It can be seen from Table 1 that the GLP-1 mutant polypeptide greatly improves its binding ability to the GLP-1 receptor, compared with GLP-1.
[0086] Table...
Embodiment 2
[0089] Example 2 Experiment of hypoglycemic function of GLP-1 mutant polypeptide in mice
[0090] Give mice intravenous injections of 8 different GLP-1 mutant polypeptides of the present invention (injection dose is 100 μg / kg), with Exendin-4 as positive control, after administration, respectively, 0.5, 4, 6, 8, 12 , Inject glucose (2g / kg) at 24 and 48 hours, measure the blood sugar content half an hour after the injection, from figure 1 It can be seen that Exendin-4 completely loses its hypoglycemic function after 4 hours, but the 8 kinds of GLP-1 mutant polypeptides still have the hypoglycemic function after 48 hours of administration.
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