Fusion polypeptides comprising GDF15 and polypeptide region capable of O-glycosylation
A technology of fusion polypeptide and glycosylation, which is applied in the direction of fusion polypeptide, medical preparations containing active ingredients, peptides, etc., can solve the problems of protein drug denaturation, difficulty in injection administration, and difficulty in drug release, so as to prolong the administration interval and reduce the Administration dose, long-lasting effect
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Embodiment 1
[0133] Example 1: Preparation of fusion polypeptides
[0134] 1.1. Preparation of fusion polypeptides comprising GDF15
[0135] Fusion polypeptides IgD-GDF15 (ID1-GDF15), IgD-IgD-GDF15 (ID2-GDF15), IgD-IgD-IgD-GDF15 (ID3-GDF15) were prepared (see Figure 2a and Figure 2b ), where the IgD hinge ( The underlined portion is the site capable of O-glycosylation) or a combination of several (1, 2 or 3) IgD hinges with GDF15 (SEQ ID NO:3, figure 1 ) fusion. To facilitate purification, a fusion polypeptide comprising a His-tag (SEQ ID NO: 15) and a TEV cleavage site (SEQ ID NO: 16) was also prepared. The amino acid sequence of each portion contained in the fusion polypeptide is summarized in Table 2 below.
[0136] 【Table 2】
[0137]
[0138] 1.1.1. Preparation of recombinant expression vector
[0139] 1.1.1.1. Mature GDF15
[0140] In order to obtain the gene encoding mature GDF15, referring to the amino acid sequence information of UniprotKB Q99968, the gene encoding ma...
Embodiment 2
[0186] Example 2. Pharmacological effects of fusion polypeptides (in vivo)
[0187] 2.1. Single Administration
[0188] 2.1.1 Test process
[0189] The pharmacological effects of the fusion polypeptides produced and purified in Example 1 above were tested in mice (C57BL / 6J, 6 weeks old, male, 100 mice; Raonbio).
[0190] In this example, a DIO mouse model (mice, C57BL / 6J-DIO, male, 100 mice, 14 weeks old (8 weeks of obese chow feeding)) was used in which C57BL / 6J mice were fed A high-fat diet was fed for 8 weeks to induce obesity. The DIO mouse model is an animal model widely used to evaluate diabetes and insulin-improving efficacy because it exhibits clinical features of type 2 diabetes such as hyperlipidemia, insulin resistance, and hyperglycemia, and is useful for metabolic diseases such as obesity , diabetes and hyperlipidemia studies have accumulated a large amount of comparable basic data, so it is suitable for the pharmacological effect test of this example, and ther...
Embodiment 3
[0246] Example 3. Pharmacokinetic testing of fusion polypeptides
[0247] 3.1. Serum preparation of test group and control group
[0248] To evaluate the pharmacokinetic profile when each fusion polypeptide was administered subcutaneously to rats, the fusion polypeptides HT-ID1-GDF15, HT-ID2-GDF15 and HT-ID3-GDF15 were administered subcutaneously in an amount of 2 mg / kg, respectively into SD rats (Koatech, male, 7 weeks old, about 250 g; n=3 each; test group), and about 200 μl of blood was collected through the tail vein at predetermined times. Blood collection times were performed before administration and at 1, 2, 4, 8, 24, 48, 72, 96, 168, 240 and 336 hours after administration. As a control group for comparison of pharmacokinetic characteristics, GDF15 (R&D Systems) was subcutaneously administered in an amount of 2 mg / kg by the same method to prepare a GDF15 administration group.
[0249] Following administration into SD rats as described above, blood collected at time p...
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