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Human insulin/analogue conjugate with continuous blood sugar reduction function and high rate of combination with receptor

A technology for recombinant human insulin and conjugates, applied in insulin, animal/human protein, hormone peptide, etc.

Active Publication Date: 2012-09-19
CHONGQING FAGEN BIOMEDICAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Novo Nordisk's long-acting insulin detemir is coupled to the 29th lysine in the B chain of insulin analogs by fatty acid myristic fatty acid (CN101784563A, CN101389650A and EP2008 / 060734), which has obvious hypoglycemic effect in vivo, and can achieve hypoglycemic effect in the human body 24 hours, but the hypoglycemic effect is only maintained within 12 hours in animals

Method used

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  • Human insulin/analogue conjugate with continuous blood sugar reduction function and high rate of combination with receptor
  • Human insulin/analogue conjugate with continuous blood sugar reduction function and high rate of combination with receptor
  • Human insulin/analogue conjugate with continuous blood sugar reduction function and high rate of combination with receptor

Examples

Experimental program
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Effect test

example 1

[0030] Example 1. Recombinant expression and purification of recombinant human insulin analog (DesB30)

[0031] In this example, according to the amino acid sequence of the human insulin analog DesB30 (Seq ID NO.2), the corresponding cDNA sequence of the yeast preferred codon is designed, which contains the leader peptide sequence EVFK and the middle short C peptide AAK corresponding to The cDNA sequence contains the restriction endonuclease Xho I and Not I sites at the 5' end and the 3' end respectively. The entire cDNA sequence is as follows (Seq ID NO.3):

[0032]CTCGAGAAGAGAGAAGTCTTCAAGTTTGTTAACCAACATTTGTGTGGTTCCCACTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTCTTCTACACTCCAAAGGCTGCTAAGGGTATCGTTGAACAATGTTGTACTTCCATCTGTTCCTTGTACCAATTGGAAAACTACTGTAACTGATAAGCGGCCGC,委托宝生物(大连)有限公司人工合成Seq ID NO.3的cDNA基因片段,并嵌入pUC18载体且命名为pUC18-DesB30。 Plasmid pUC18-DesB30 and expression vector plasmid pICZαA were double-digested with restriction endonucleases Xho I and Not I, recovered by gel, ligated ...

example 2

[0037] Example 2. Preparation of recombinant human insulin analogue conjugate (DesB30-5K)

[0038] Take 100ml of recombinant human insulin analog (DesB30) solution (containing 20mmol / L Tris-HCl) with a concentration of 2mg / ml, adjust the pH to 10.5, stir at 100rpm at room temperature, first add mPEG-SPA ( The molecular weight is 5K, purchased from Beijing Jiankai Technology Co., Ltd.), after reacting for 30 minutes, add mPEG-SPA at a molar mass ratio of 1:1.5, continue to react for 30 minutes, and adjust the pH to 3.0 with 1% trifluoroacetic acid to terminate the reaction , The modified sample was subjected to reverse-phase preparative C18 column chromatography to separate and remove unmodified DesB30, free PEG molecules, and other conjugates not modified at the B29 position. The separation conditions are: liquid A is 0.1% trifluoroacetic acid and 5% acetonitrile, liquid B is 0.1% trifluoroacetic acid and 95% acetonitrile, the DesB30-5K conjugate is collected by linear gradien...

example 3

[0039] Example 3. Preparation of recombinant human insulin analogue conjugate (DesB30-10K)

[0040] Take 100ml of recombinant human insulin analogue (DesB30) solution (containing 20mmol / L Tris-HCl) with a concentration of 2mg / ml, adjust the pH to 10.5, stir at 100rpm at room temperature, first add mPEG-SPA ( The molecular weight is 10K, purchased from Beijing Jiankai Technology Co., Ltd.), after reacting for 30 minutes, add mPEG-SPA at a molar mass ratio of 1:2, continue to react for 30 minutes, adjust the pH to 3.0 with 1% TFA to terminate the reaction, modify Afterwards, the sample was subjected to reverse-phase preparative C18 column chromatography to separate and remove unmodified DesB30, free PEG molecules, and other conjugates not modified at the B29 position. The separation conditions are: liquid A is 0.1% trifluoroacetic acid and 5% acetonitrile, liquid B is 0.1% trifluoroacetic acid and 95% acetonitrile, the DesB30-10K conjugate is collected by linear gradient elution...

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Abstract

The invention relates to a human insulin / analogue conjugate with a continuous blood sugar reduction function and a high rate of combination with a receptor, and discloses a method for preparing a reconstructed human insulin / analogue conjugate by reconstructing a human insulin precursor by utilizing a methylotrophic yeast expression, performing purification, enzyme digestion, chromatography or synthesis to prepare a reconstructed human insulin and a DesB30 analogue thereof and coupling a side chain amino group of a 29th-bit lysine of a B chain of the reconstructed human insulin precursor and a polyethyleneglycol acetamide bond. The prepared reconstructed human insulin conjugate has the characteristics of high rate of combination with the insulin receptor, remarkable continuous blood sugar reduction effects and long in-vivo half-life period, and is applied to the treatment of I-type and II-type diabetes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to the recombinant preparation, modification, purification and application of human insulin and analogues. Background technique [0002] Insulin is a protein secreted by pancreatic beta cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. It contains 51 amino acids and has a molecular weight of about 5800. Insulin The molecule has an A chain (21 amino acids) and a B chain (30 amino acids) bound by two disulfide bonds. If the disulfide bond is opened, it will lose its activity. B cells first synthesize a macromolecule of pre-proinsulin, which is then processed into proinsulin of octahexapeptide, which is then hydrolyzed into insulin and connecting peptide. The main physiological function of insulin is to regulate the metabolic process: 1. Promote the uptake and utilization of glucose by tissue ce...

Claims

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Application Information

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IPC IPC(8): C07K14/62A61K47/48A61K38/28A61P3/10
CPCA61K47/48215A61K38/28C07K14/62A61K47/60A61P3/10
Inventor 范开曹宇陈勇李红亮陈海容万颖
Owner CHONGQING FAGEN BIOMEDICAL
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