Polyethylene glycol-modified exendin analog, and preparation method and application thereof

[0099] Example 1. Preparation of linear polyethylene glycol modified Ex4-Cys (C40-PEG-Ex4-Cys)

[0100] A polypeptide (Ex4-Cys) with the following sequence was synthesized using standard polypeptide solid-phase synthesis technology:

[0101] His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser-Cys; (Seq ID No.1)

[0102]3, 5, 10, 20 and 50 kDa polyethylene glycols with maleine groups (Nektar, mPEG-pro-pionalaldehyde, mPEG-ALD, 2 kDa, 0.95 mg / ml in 50 mM sodium acetate, pH 5.5) Add respectively 0.5mL Exendin-4 (1 mg / ml in 50mM sodium acetate, pH 5.5), and then add 20mM NaC-NBH3 as reducing agent. The molar ratio of mPEG-ALD to Ex4-Cys is 1:1-2. mPEG-ALD and Ex4-Cys were reacted at 4°C for 2 hours under dark conditions. The reaction was terminated with 0.1% aqueous trifluoroacetic acid (TFA) to obtain PEG-modified Ex4-Cys, named as C40-PEG-Ex4-Cys.

[0103] Example 2. Preparation of branched polyethylene glycol modified EX4-Cys (C40-tPEG-Ex4-Cys)

[0104] Add 0.5 mL Ex4-Cys (1 mg / ml in 50 mM sodium acetate, pH 5.5), then 20 mM NaC-NBH 3 as a reducing agent. The molar ratio of mPEG-ALD to Ex4-Cys is 1:1-2. mPEG-ALD and Ex4-Cys were reacted at 4°C for 2 hours under dark conditions. The reaction was terminated with 0.1% aqueous trifluoroacetic acid (TFA) to obtain branched PEG-modified Ex4-Cys with different molecular weights, named C40-tPEG-Ex4-Cys. Purified C40-tPEG-Ex4-Cys as figure 1 Shown, its retention time is 18.5 minutes.

[0105] Example 3. Analysis of the influence of PEG modification on the physiological activity image of Ex4-Cys

[0106] To detect Exendin-4, PEG-modified Ex4-Cys of different molecular weights reacted with the GLP-1 receptor at a density of 2.5×10 5 The insulin-secreting cells INS-1 were seeded on a 12-well plate, 10 per well 5 cells, and then cultured for 2 days to allow them to stably attach to the bottom of the culture plate. After the cells adhered to the wall, labeled with 125 The buffer of I-Exendin-4 (Exendin-4 derivative extending from amino acid residue 9 and amino acid residue 39) was used to replace the cell culture medium, and a certain amount of buffer was added to form a final concentration of 30 μM. Thereafter, a certain amount of natural Exendin-4 and PEG-modified Ex4-Cys of different molecular weights were added to form a final concentration of 0.001-1000 nM, and incubated at room temperature for 2 hours to enable it to competitively bind to the receptor. Wash