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Recombination preparation method and application of Exendin-4 derivative

A technology of derivatives and recombinant plasmids, applied in the field of bioengineering, can solve the problems of reduced immunogenicity stability, unfavorable industrial production, difficult separation and purification of products, etc. The effect of prolonging the potency of the drug

Inactive Publication Date: 2010-03-10
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The American Amylin company uses one or more polyethylene glycol (PEG) polymers to modify the ε-amino group on the Exendin-4 lysine. Compared with the natural Exendin-4, the above-mentioned Exendin-4 derivatives can significantly prolong the half-life. In addition, It can also reduce immunogenicity, resist protease hydrolysis, improve bioavailability, reduce toxicity and increase stability, but the preparation process of this method is more complicated, the product separation and purification are difficult, and the yield is very low, which is not conducive to industrial production
[0005] Exendin-4 derivatives can be prepared by solid-phase chemical synthesis, see the inventor's invention patent application (application number is "200910045187.6", the title of the invention is "an Exendin-4 derivative and its solid-phase chemical synthesis"), But the cost of this preparation method is relatively high, therefore, the present invention provides a kind of cost-effective, utilizes the method for preparing Exendin-4 derivative by DNA recombination technology

Method used

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  • Recombination preparation method and application of Exendin-4 derivative
  • Recombination preparation method and application of Exendin-4 derivative
  • Recombination preparation method and application of Exendin-4 derivative

Examples

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Effect test

Embodiment 1

[0031] Example 1 The Exendin-4 derivative of the present invention was prepared by DNA recombination technology. The molecular structural formula of the derivative is Seq ID No.2, that is, Exendin-4(1-39)-Xaa40, wherein Xaa40=Cys;

[0032] In step (1), according to the amino acid sequence of the Exendin-4 derivative, select Escherichia coli preferred codons, and synthesize the following fragment with a length of 164bp by PCR method, namely Seq ID No.9:

[0033] gccctagatc tggatgacga tgacaagcat ggcgaaggca cctttaccag cgatctgagc 60

[0034] aaacagatgg aagaagaagc ggtgcgcctg tttattgaat ggctgaaaaa tggtggtccg 120

[0035] agcagcggtg cgccgccgcc gagcnnntaa tgaaagctta aact 164

[0036] Wherein, n145=t, n146=g, n147=c;

[0037] Wherein, the fragment contains an enterokinase EK site, an Exendin-4 derivative gene, a stop codon TAA, and restriction endonuclease BglII and HindIII sites;

[0038]In step (2), the above-mentioned gene fragment is double-digested with restriction endonuclease...

Embodiment 2

[0043] Example 2 Preparation of Exendin-4 derivatives of the present invention by DNA recombination technology, the molecular structural formula of the derivatives is Seq ID No.3, namely Exendin-4(1-39)-Xaa40-Xaa41, wherein Xaa40=Cys, Xaa41=Gly ;

[0044] In step (1), according to the amino acid sequence of the Exendin-4 derivative, select Escherichia coli preferred codons, and synthesize the following fragment with a length of 167bp by PCR method, namely Seq ID No.10:

[0045] gccctagatc tggatgacga tgacaagcat ggcgaaggca cctttaccag cgatctgagc 60

[0046] aaacagatgg aagaagaagc ggtgcgcctg tttattgaat ggctgaaaaa tggtggtccg 120

[0047] agcagcggtg cgccgccgcc gagcnnnnnn taatgaaagc ttaaact 167

[0048] Wherein, n145=t, n146=n148=n149=g, n147=n150=c;

[0049] Wherein, the fragment contains an enterokinase EK site, an Exendin-4 derivative gene, a stop codon TAA, and restriction endonuclease BglII and HindIII sites;

[0050] All the other steps are the same as in Example 1.

Embodiment 3

[0051] Example 3 Preparation of Exendin-4 derivatives of the present invention by DNA recombination technology, the molecular structural formula of the derivatives is Seq ID No.3, namely Exendin-4(1-39)-Xaa40-Xaa41, wherein Xaa40=Gly, Xaa41=Cys ;

[0052] In the Seq ID No.10 gene fragment synthesized in step (1), n145=n146=n149=g, n147=n150=c, n148=t.

[0053] All the other steps are the same as in Example 1.

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Abstract

The invention relates to a recombination preparation method and an application of an Exendin-4 derivative with Exendin-4 activity. The molecular structural formula of the derivative is one of the following six forms of Exendin-4 (1-39)-Xaa40 (Seq ID No.2), Exendin-4 (1-39)-Xaa40-Xaa41 (Seq ID No.3), Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42 (Seq ID No.4), Exendin-4 (1-30)-Xaa31-(Seq ID No.6), Exendin-4(1-30)-Xaa31-Xaa32 (Seq ID No.7) and Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33 (Seq IN No.8), wherein Xaa40, Xaa41, Xaa42, Xaa31, Xaa32 and Xaa33 are respectively any one amino acid of Cys, Ala, Gly, His, Serand Thr. The derivative is prepared by a DNA recombination technology, has a longer half-life period than Exendin-4, is easy to purify with low cost, and is especially suitable to be used as an activecomponent of the medicament in the preparation of a medicament for treating diabetes.

Description

technical field [0001] The invention relates to an Exendin-4 derivative with Exendin-4 activity and its preparation and application, belonging to the technical field of bioengineering. Background technique [0002] In 1992, Eng et al. isolated a protein consisting of 39 amino acids from the saliva of the Gila monster (Heloderma suspectum) distributed in Arizona and northern Mexico, which had 53% homology with GLP-1. It was named Exendin-4. Subsequent studies have shown that Exendin-4 and GLP-1 act on the same receptor, have similar signal transduction pathways, and have exactly the same physiological functions in mammals. Exendin-4 can stimulate glucose-dependent insulin secretion, that is, it stimulates insulin secretion when the blood sugar concentration is high, and does not stimulate insulin secretion at low and normal concentrations; it also has the functions of promoting β-cell proliferation and regeneration, inhibiting glucagon secretion, and slowing down the secreti...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02A61K38/17A61P3/10C12R1/19
Inventor 汪洋冯震顾娟吴自荣黄静劳勋
Owner EAST CHINA NORMAL UNIVERSITY
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