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Monomer quick-acting insulin as well as preparing method and application thereof

A technology of insulin and monomers, which is applied in the fields of medicine and pharmacy, and can solve problems such as excess, porcine insulin shock, and withdrawal from the market

Inactive Publication Date: 2010-09-01
费俭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the emergence and application of genetically engineered human insulin in the past ten years, the market for porcine insulin has been greatly impacted, and there is a surplus. In the end, it may be completely replaced by recombinant human insulin and withdraw from the market.

Method used

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  • Monomer quick-acting insulin as well as preparing method and application thereof
  • Monomer quick-acting insulin as well as preparing method and application thereof
  • Monomer quick-acting insulin as well as preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Genetic engineering to express B22Glu-desB30 insulin

[0109] In this example, the C-terminus of the B chain of B22Glu-desB30 insulin is connected to the N-terminus of the A chain with a linker peptide (such as Ala-Ala-Lys) to form an expression precursor (EPIP), because the structure of the precursor is conducive to the expression of insulin in After biosynthesis, it folds and forms the correct disulfide bonds. Proceed as follows:

[0110] The precursor gene was obtained by DNA synthesis method, and the sequence is as follows;

[0111] ttc gtt aac caa cac ttg tgc ggt tcc cac ttg gtt gag gct ttg tac ttg gtt tgc ggt gaa gaa ggt ttc t tc tac act cct aag gct gct aag ggt att gtcgaa caa tgc tgt acc tcc atc tgc tcc ttg tac caa ttg gaa aac tac tgc aac (SEQ ID NO: 3)

[0112] After adding BamH1 and EcoR1 restriction sites at both ends, the BamH1 and EcoR1 restriction sites were used to clone into pPIC9K (purchased from INVITROGEN) plasmid to obtain pPIC9K / EPIP. pPIC9K / EPIP...

Embodiment 2

[0116] Determination of self-polymerization properties of dezincified insulin

[0117] In this example, the self-polymerization properties of dezincified insulin were determined by conventional methods.

[0118] Higher concentrations of insulin in neutral solution exist as a mixture of monomers, disomes, and hexasomes. Chromatography with molecular sieves: Superdex 75 (HR 10 / 30) column, mobile phase is phosphate buffer, pH 7.4, flow rate is 0.5ml / min, sample volume is 40 microliters, room temperature, 230nm detection, peptide concentration from Low to high are: 38μM (0.22mg / mL), 75μM (0.43mg / mL), 150μM (0.86mg / mL), 300μM (1.73mg / mL), 600 (3.46mg / mL)μM. The average molecular weight of the mixture is described by the partition coefficient Kd. Kd=(Vr-V 0 ) / (Vc-V 0 ), where Vr is the outflow volume, V 0 is the external water volume, and Vc is the total column bed volume.

[0119] The results show that with the increase of dezincified insulin concentration, its retention time...

Embodiment 3

[0123] Determination of Self-polymerization Properties of B22Glu-des-B30 Insulin by Molecular Sieve Column Chromatography

[0124] In this example, the self-polymerization property of the monomeric insulin prepared in Example 1 was measured by conventional methods.

[0125] Except the polypeptide concentration, the implementation conditions are the same as in Example 2. The peptide concentration is: 2mg / ml, 5mg / ml, 10mg / ml. With the increase of the concentration of the peptide, the peak time, peak shape and Kd value of B22Glu-desB30 insulin do not change with the change of the peptide concentration ( Figure 5 ,Table 2). This indicated that B22Glu-desB30 insulin exists in the form of monomer at the injection concentration of 500μM (3mg / ml).

[0126] Table 2B22Glu-desB30 insulin

[0127] Kd value on molecular sieve column Superdex 75 (HR 10 / 30) column chromatography

[0128]

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Abstract

The invention relates to a new insulin B chain, monomer quick-acting insulin and a medical composition which comprises the monomer quick-acting insulin. The invention also relates to a preparing method and application of the monomer quick-acting insulin.

Description

[0001] This application is a divisional application filed on April 29, 2006, and the application number is CN200610026236.8. technical field [0002] The present invention relates to the fields of medicine and pharmacy. More specifically, the present invention relates to novel insulin B chains, novel monomeric rapid-acting insulins, and pharmaceutical compositions containing the monomeric rapid-acting insulins. The invention also relates to a method for preparing the monomeric fast-acting insulin. Background technique [0003] The incidence of diabetes is increasing year by year, and it can cause death from cardiovascular disease. It is one of the major diseases threatening human health today. According to the results of the United Kingdom Prospective Diabetes Study, the treatment of diabetes is aimed at implementing fine blood sugar control to avoid diabetic syndrome. Although insulin is commonly used in type 1 diabetes, recent studies have shown that one-third of people ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C12P21/02A61K38/28A61P3/10
Inventor 张友尚费俭朱尚权石嘉豪都海娟崔大敷陆怡
Owner 费俭
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