Polypeptide and application thereof

An amino acid and N-terminal technology, applied in the field of biomedicine, can solve problems such as short half-life, increased medical expenses, and inability to completely reverse islets, and achieve high stability effects

Inactive Publication Date: 2021-03-30
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this type of drug has a short half-life and has adverse reactions in the digestive tract such as nausea and vomiting.
Although the above drugs can play a hypoglycemic effect, they cannot complet...

Method used

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  • Polypeptide and application thereof
  • Polypeptide and application thereof
  • Polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Recombinant expression plasmid PET28A-BEFA, PET28A-MBP-B construction

[0085] 1, β cell proliferative factor A (BEFA), all gene synthesis of MBP partner protein

[0086] The person literature (Jennifer Hampton Hill, A conserved bacterial protein inducespancreatic beta cell expansion during zebrafish development, elifesciences, Dec.2016) gut BefA published amino acid sequence (co 258aa) secreted by strain Enterococcus gallinaru, according to E. coli (BL21 ( The optimization principle of DE3)) is optimized for nucleic acid sequences. At the same time, the sequence is strictly defined, and the sequence 3 'end adds a termination codon (TAA) to obtain the encoding gene BEFA, and finally handed over to the gene synthesis company for all gene synthesis (PUC57-BEFA).

[0087] The MBP partner gene is encoded containing a TEV (cysteine ​​protease) enzyme disaster in TEV (tobacco corrusor virus), and the nucleic acid sequence is optimized according to the optimization princ...

Embodiment 2

[0092] Example 2 Engineering Cell Expression Production BEFA / Befa Mutant

[0093] 1, shake flask fermented befa / mbp-befa protein

[0094] The positive strain glycerol tube obtained by screening and identifying 0.2% inoculated to 5 ml of 50 μg / ml kanamycin LB liquid medium, 37 ° C 250 rpm activated overnight; inoculated by 2% inoculation to 50 μg / ml In the 2000ml triangular bottle of kanamycin LB liquid medium, the amount of liquid is 20%, 37 ° C 250 rpm to OD 600 When = 0.6 ~ 0.8, IPTG was added to 1 mm, and the final concentration was 1 mM, and the expression of 4 to 6 h was induced at 37 ° C; the fermentation broth was centrifuged (8000 rpm 10 min), and the bacteria was collected. After the purified water resuspended, the bracket (ultrasonic crushing or homogenization is broken), and the fragmentation is purified by low temperature high speed (12000 rpm 30min 4 ° C), and the supernatant is obtained.

[0095] 2, purify the Befa protein

[0096] BEFA protein uses nickel pi...

Embodiment 3

[0120] Example 3 BEFA and BEFA mutant activity detection

[0121] 1, INS-1 cell proliferation experiment

[0122] INS-1 (rat inside islet cell tumor cell) cells Press 3 * 10 4 ~ 6 * 10 4 Cells / ML, Press 100 μL / Well to 96-well plate, 37 ° C, 5% CO 2 Culture 48h. Exchange to sugar-free 1640 + 0.1% BSA, hunger 24h. Discard the hunger medium, add 1640 + 1% FBS medium dilution to 10 -9 M ~ 10 -7 M'S BEFA Protein (or Befa Mutant Befa Modification) Stimulation, 100μL / Well. 37 ° C, 5% CO 2 Culture 72h. Add CCK8 reagent, 10 μL / Well, 37 ° C, 5% CO 2 After 2 hours of incubator, detection OD 450 . See Figure 7-8 . BEFA proteins and BEFA mutants can significantly promote proliferation of INS-1 cells.

[0123] 2, mouse pharmacodynamic experiment

[0124] (1) BEFA protein OB / ob mouse pharmacodynamic experiment

[0125] Ob / OB mouse divided by 8 animals per group, 30 mg / kg of Befa protein and PBS buffer per group, 1 time per day, 4 weeks of administration, oral sugar at the 7th day ...

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Abstract

The invention provides a polypeptide. The polypeptide has a modification at the N-terminus compared to a polypeptide having an amino acid sequence selected from at least one of addition, methylation,formylation, carbamylation, succinylation, cyclization, propionylation, hexadecane acylation, tetradecane acylation and acetylation. The polypeptide is: (1) a polypeptide having an amino acid sequencerepresented by SEQ ID NO: 1-6; or (2) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity as compared to (1); or (3) a polypeptide having substitution, deletion and/or addition of one or more amino acids as compared to (1). According to the polypeptide disclosed by the embodiment of the invention, the activity of the BefAprotein for promoting beta cell proliferation is maintained, the polypeptide has the characteristics of higher stability and difficulty in degradation, and the polypeptide has the potentials of reducing blood glucose and weight and protecting the liver.

Description

[0001] Priority information [0002] The present invention is submitted to the China National Intellectual Property Office on January 2, 2020, and the application name CN202010003240.2, the application name is "polypeptide and its application", and its entire content is incorporated herein by reference this invention. Technical field [0003] The present invention relates to the field of biomedicine, and Compositions and their application in the field of metabolic diseases such as diabetes. Background technique [0004] The lack of insulin secretion cells has always been considered to be one of the etiology of type I diabetes. In type I diabetes, the immune system incorrectly attacks and destroy β cells. In recent years, researchers have found that lack of functional beta cells are also an important cause of Type II diabetes, and the advanced islet function of Type II diabetes is gradually declined. Therefore, it is the main focus of diabetes study by the development of drugs that...

Claims

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Application Information

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IPC IPC(8): C07K14/315C07K1/107C07K19/00C12N15/31C12N15/62A61K38/16A61P3/10A61P3/04A61P1/16
CPCC07K14/315A61P3/10A61P3/04A61P1/16A61K38/00C07K2319/24C07K2319/50
Inventor 李利佳古想娣吴都郭林峰李晓平胡育龙李宇晟李玉许玲华李静陈小锋李文佳
Owner SUNSHINE LAKE PHARM CO LTD
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