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Fusion protein and application thereof

A fusion protein and active protein technology, applied in the field of fusion protein and pharmaceutical composition containing the fusion protein, can solve the problems of short half-life in vivo, loss of part or all activity, poor drug stability, etc.

Pending Publication Date: 2021-10-22
博锐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, biological drugs also have some outstanding problems, such as imperfect drug delivery system, poor drug stability, short half-life in vivo and low bioavailability, etc.
Those skilled in the art sometimes optimize the metabolic kinetics of biological drugs through modification to achieve the purpose of prolonging the half-life. However, not all drugs can be modified, and some drugs will lose part or all of their activity during the modification process.

Method used

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  • Fusion protein and application thereof
  • Fusion protein and application thereof
  • Fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0212] Embodiment 1: Preparation of the fusion protein shown in SEQ ID No:9-12

[0213] The fusion protein involved in this application (for example, SEQ ID No: 9-12) can be prepared by fermentation using Escherichia coli as a host cell. The cDNA sequence encoding the fusion protein was ligated into the pET15b plasmid through enzyme digestion, and then transfected into Escherichia coli. The obtained positive clones were subjected to primary fermentation, and then the primary fermentation liquid was moved to a secondary fermenter (500 L), and IPTG induction was performed when the OD value was 2-5. After protein expression, the mycelia were collected and subjected to cell disruption. After the broken mycelia was collected and resuspended, it was purified with a C 8 liquid phase column and lyophilized for future use.

Embodiment 2

[0214] Embodiment 2: the stability of the fusion protein as shown in SEQ ID No:9

[0215] Diabetic rats were injected with GLP-1 (control group) or the fusion protein shown in SEQ ID No:9 (dosage is 0.1M / kg bw), and the ophthalmic plexus vein was performed at different time points after injection. About 0.2 mL of blood was collected to prepare serum for use.

[0216] The blood concentration of GLP-1 in rat serum was detected by enzyme-linked immunosorbent assay (ELISA), and the operation was as follows: centrifugation at 4° C., and centrifugation at 13,000 rpm / min for 20 minutes to obtain serum. The serum was incubated with 100 mM ammonium acetate at room temperature for 10 minutes, and the concentration of GLP-1 was determined with the GLP-1 EIA kit. For the test method, refer to the instructions of the kit and evaluate the stability of GLP-1 according to the results. The result is as figure 1 shown.

[0217] The results showed that the fusion protein prolongs the stabili...

Embodiment 3

[0218] Example 3: Long-acting hypoglycemic function of the fusion protein as shown in SEQ ID No:10

[0219] Each group of 10 rats suffering from diabetes is grouped, and the oral glucose stimulation of 2g / kg body weight is carried out respectively, followed by oral administration of insulin or the fusion protein shown in SEQ ID No:10 (experimental dosage is 0.5mM insulin / kg bw or 0.1mM fusion protein / kg bw). Detect changes in animal blood sugar levels, see the results figure 2 . The results showed that the route of oral administration of insulin made the insulin rapidly degraded and inactivated in the stomach without drug effect. However, the fusion protein of the present application can protect insulin from acidic environment damage or intestinal protease degradation, and the size of the fusion protein meets the requirements of intestinal absorption (60-120nm).

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PUM

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Abstract

The invention provides a fusion protein. The fusion protein comprises an active protein, a linker and a polypeptide, wherein the polypeptide comprises S1 and S2, and the S1 and the S2 are connected in series through a peptide bond in the direction from the N end to the C end; S1 is an amino acid sequence as shown in SEQ ID No: 1 or a functional variant of the amino acid sequence; and S2 is an amino acid sequence as shown in SEQ ID No: 7 or a functional variant of the amino acid sequence.

Description

technical field [0001] The present application relates to the field of medicine preparation, more specifically, the present application relates to a fusion protein, a pharmaceutical composition comprising the fusion protein and uses thereof. Background technique [0002] Biological drugs (especially nucleic acid or protein drugs) are widely used to treat many diseases, such as cancer, autoimmune diseases, and AIDS. However, biological drugs also have some outstanding problems, such as imperfect drug delivery system, poor drug stability, short half-life in vivo, and low bioavailability. Those skilled in the art sometimes optimize the metabolic kinetics of biological drugs through modification to achieve the purpose of prolonging the half-life. However, not all drugs can be modified, and some drugs will lose part or all of their activity during the modification process. Therefore, there is an urgent need to find new drug fusion proteins in this field. [0003] Summary of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/28A61K38/26A61K47/68A61P3/10
CPCC07K14/62C07K14/605C07K14/505C07K14/71C12N15/63A61P3/10C07K2319/00A61K38/00
Inventor 龚珉
Owner 博锐生物科技有限公司