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Preparation method of human insulin analogue and usage thereof

A technology of human insulin and analogs, applied in the field of human medicine, can solve the problems of inconvenience of patients, unable to effectively simulate the physiological secretion of insulin after meals, unable to effectively simulate the physiological secretion of insulin and so on.

Inactive Publication Date: 2008-04-09
江苏未名生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitations of the action properties of natural human insulin after subcutaneous or intramuscular injection immediately before meals, it cannot effectively simulate the physiological secretion of postprandial insulin, so insulin therapy cannot be effectively performed (Dewitt DE, et al., JAMA 2003, 289:2254; Binder C., et al., New York: Raven Press, 1983, 6:53.)
[0005] Immediate subcutaneous or intramuscular injection of natural human insulin before meals cannot effectively simulate the physiological secretion of postprandial insulin, and injection of natural human insulin half an hour before meals brings great inconvenience to patients' lives. Insulin preparations for physiological prandial insulin secretion curves - rapid-acting insulin analogs

Method used

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  • Preparation method of human insulin analogue and usage thereof
  • Preparation method of human insulin analogue and usage thereof
  • Preparation method of human insulin analogue and usage thereof

Examples

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Effect test

preparation example Construction

[0054] The preparation method of this human insulin analog is completed by the following steps:

[0055] (1) construct a kind of reproducible expression vector, this vector comprises a section of DNA sequence encoding the human insulin analogue with amino acid sequence II:

[0056] Met-X 2 b -Y-R 3 -R 2 -R 1 -Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-

[0057] Leu-Tyr-leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-X 1 a -Arg-Gly-

[0058] Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

[0059] II

[0060] where X 2 b is a peptide chain containing b amino acid residues, b is a non-negative integer; X 1 a is a peptide chain containing a amino acid residues, a is a non-negative integer; Y is Arg or Lys; R 1 Any amino acid residue in Pro, Ala, Gly, Ser, Val, Leu; R 2 is any desired genetically codable amino acid residue; R 3 It is an n segment-R2-R1-dipeptide fragment, n ta...

Embodiment 1

[0081] Example 1 Construction of FPPIns Recombinant Genetic Engineering Bacteria

[0082] 1. Acquisition of PPIns gene

[0083] Two primers P1 and P2 were designed by means of computer software, and the nucleotide sequences of the two primers were as follows:

[0084] P1: 5'ACAGGATCCAAGCGTCCGAAACCGTTTGTCAATCAGCACCTT 3'

[0085] P2: 5'TTAAAGCTTAGTTGCAGTAGTTCTCC 3'

[0086] A BamH I restriction site was introduced into primer P1, and a Himd III restriction site was introduced into primer P2. Primers were synthesized by Shanghai Yingjun Company.

[0087] Using the pET28a-ProIns plasmid as a template, see Figure 1, and using P1 and P2 as upstream and downstream primers to perform PCR amplification to obtain the PPIns gene fragment.

[0088] PCR reaction system: 100 pmol P1, 100 pmol P2, 2 μl dNTP (10 nM), 5 units of PfuDNA polymerase, 0.5 μl of pET28a-ProIns plasmid, 5 μl 10x Pfu DNA polymerase reaction buffer, the total system is 50 μl.

[0089] PCR reaction conditions: 94°C...

Embodiment 2

[0094] Example 2 Expression of FPPIns gene in Escherichia coli

[0095]Pick a single colony from the culture plate of the pET28a-FPPIns recombinant genetically engineered bacteria, inoculate it in LB liquid medium containing 50 μg / ml kanamycin, culture it overnight at 37°C with constant temperature shaking, and inoculate it at a ratio of 1%. In LB liquid medium (50 μg / ml kanamycin), after culturing at 37°C for 4 hours, α-lactose at a final concentration of 0.5 mmol / L was added to induce Escherichia coli to express T7 RNA polymerase, and the culture was continued to express the fusion protein FPPIns. After 6 hours of induction, the cells were recovered by centrifugation, and SDS-PAGE electrophoresis and thin-layer scanning showed that the expression of the FPPIns precursor proinsulin protein gene had been realized, and the expressed protein accounted for about 40% of the total bacterial protein. The results are shown in Figure 4.

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Abstract

The present invention pertains to the field of human medicine, which relates to a drug for the treatment of type I and type II diabetes and provides a preparation method and the usage of a human insulin analogue. The human insulin analogue of the present invention is characterized in that the human insulin analogue has the form as the right formula, wherein, R1 is any one amino acid residue of Pro, Ala, Gly, Ser, Val and Leu; R2 is any needed amino acid residue which can be encoded genetically; R3 is n paragraphs of R2-R1-dipeptide fragment, n is any one number of 0, 1, 2, 3 and 4. The human insulin analogue of the present invention is prepared by using a biological engineering method, the active insulin analogue has the advantages that the human insulin analogue is not easy to from the dimer and mainly exists by a monomer form after the subcutaneous injection, the local absorption is faster, the onset time is shorter, and the pharmacokinetics characteristics are more in line with the change spectra of physiological insulin and blood glucose.

Description

technical field [0001] The invention belongs to the field of human medicine, and in particular relates to a kind of medicine for treating type I and type II diabetes. More specifically, it relates to a preparation method and application of a class of human insulin analogues. Background technique [0002] About 120 million people in the world suffer from diabetes, of which about 12 million people are type 1 diabetes. For these people, the use of exogenous insulin to replace the lack of endocrine insulin is currently the only feasible therapy. Hyperglycemia is the cause of various diabetic complications, so it is necessary to strictly control the daily 24-hour blood glucose concentration within the normal range to prevent or delay the late complications of diabetes. [0003] The physiological secretion of insulin in a normal human body is regulated by blood sugar concentration. Generally, the blood insulin concentration reaches its peak value 30-60 minutes after a meal, and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62A61K38/30A61P3/10A61P5/48C12N15/09C12N15/12
Inventor 刘景晶鲁勇刘素丽李祝芳范豪吴洁曹荣月
Owner 江苏未名生物医药有限公司
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