Acylated long-acting GLP-1 derivative
A technology of GLP-1 and derivatives, which is applied in the field of peptide technology and its derivatives, can solve the problems of limiting action time, etc., and achieve the effects of excellent hypoglycemic ability, significant weight loss ability, and excellent weight loss ability
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Embodiment 1
[0070] Example 1 Preparation of Acylated Long-acting GLP-1 Derivatives
[0071] This example provides a variety of long-acting GLP-1(7-37) derivatives and their preparation methods, especially the construction of a recombinant engineering bacterium that can efficiently express the derivatives of the present invention. The preparation method is as follows (using HS-Y1 as the example):
[0072] (1) Build the code [Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]-GLP-1(7-37) expression plasmid
[0073] Through a large amount of research and experiments in the early stage, the present invention selects FKFEFKFE as the inclusion body-promoting sequence and DDDDK as the EK restriction sequence, and fuses the inclusion body-promoting sequence, the EK restriction sequence and the GLP-1 analog coding gene sequence in series in turn to obtain the following sequence: The coding gene fragment shown in SEQ ID NO.3; through the NdeI and XhoI sites, the above-mentioned fragment was inserted ...
Embodiment 2
[0094] Example 2: In vitro cell affinity activity assay
[0095] (1) Preparation of different long-acting GLP-1 derivatives for injection
[0096] The specific formula is: DMEM blank medium, GLP-1 derivatives: 320nM, 64nM, 12.8nM, 2.56nM, 0.512nM, 0.1024nM, 0.02048nM, 0.004096nM. Among them, positive control group (semaglutide), experimental group 1 (HS-Y1), and experimental group 2 (HS-Y2).
[0097] Select HEK293 / Luc / GLP1R cells in good culture condition, discard the culture medium in the bottle, wash once with PBS buffer, add 0.05% Trypsin digestion solution to digest for 3 minutes, then add DMEM basal medium to stop digestion, and collect cells by centrifugation. Adjust the cell density to 8.0 × 10 with DMEM blank medium 5 cells / mL, 50 μL / well were inoculated in 96-well cell culture plates, at 37°C, 5% CO 2 Incubate overnight under conditions.
[0098] Use Fire-Lumi luciferase assay kit to detect the in vitro activity of derivatives of GLP-1 analogs: prepare the assay m...
Embodiment 3
[0104] Example 3: Study of hypoglycemic effect in mice
[0105] (1) Experimental materials:
[0106] a. Experimental drug preparation:
[0107] Prepare injections of different GLP-1 derivatives, the specific formula is 1.133mg / mLNa 2 HPO 4 , 5.5mg / mL phenol, 14.0mg / mL propylene glycol, and GLP-1 derivatives;
[0108] b. Animal experiments:
[0109] Fifty healthy SPF male KM mice aged 6-8 weeks and weighing 18-20g were selected and divided into blank control group, model control group, positive control group (semaglutide group), Example 1 group, Example 1 2 groups of which:
[0110] Blank control group: including 10 mice, gavage with pure water, and subcutaneous injection of blank solvent in the abdomen;
[0111] Model control group: including 10 mice, intragastric administration of glucose (4g / kg), and subcutaneous injection of blank in the abdomen;
[0112] Positive control group: including 10 mice, intragastric administration of glucose (4g / kg), and subcutaneous injec...
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