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Exendin-4 analogue, preparation method thereof and application Exendin-4 analogue

A technology of analogs and amino acids, applied in the field of genetic engineering in the Ming Dynasty, can solve the problems of not being suitable for large-scale production and preparation, and the price of cutting enzymes is expensive

Inactive Publication Date: 2013-06-05
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Due to the small molecular weight of Pro-Pro-Exendin-4-Asp, it is often easily degraded by enzymes in bacterial cells during the synthesis process of genetic engineering methods. In genetic engineering, this small peptide is usually linked to a fusion partner with a slightly larger molecular weight. Co-expression is achieved, and the fusion partner is separated from the target peptide with thrombin or enterokinase after expression, but this cutting enzyme is expensive and not suitable for large-scale production preparation

Method used

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  • Exendin-4 analogue, preparation method thereof and application Exendin-4 analogue

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Effect test

Embodiment 1

[0046] The cloning of embodiment 1Pro-Pro-Exendin-4-Asp gene

[0047] In this experiment, overlapping PCR technology was used, and 4 primers M1, M2, M3, and M4 with complementary ends were used to make the PCR products form overlapping chains, so that in the subsequent amplification reaction, amplified genes from different sources were extended. The augmented fragments are overlapped and spliced ​​together to form the gene sequence of Pro-Pro-Exendin-4-Asp. The sequences of the four primers are as follows:

[0048] M1: 5′-ATAGGATCCACCACATGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAG-3′

[0049] M2: 5′-CAATAAACAGACGCACCGCTTCTTCTTCCATCTGTTTGCTCAGATCGCTG-3′

[0050] M3: 5′-GCTTGGACCACCGTTTTTCAGCCATTCAATAAACAGACGCACCGC-3′

[0051] M4: 5′-TATAAGCTTAATCGCTTGGTGGTGGTGCACCGCTGCTTGGACCACCGTTTTTC-3′

[0052] Primers were synthesized by Jerry Sequencing Company. The PCR reaction was carried out on an eppendorf PCR amplification instrument. Among them, M1, M2, M3, and M4 were mixed according...

Embodiment 2

[0054] Expression and cultivation of embodiment 2 Pro-Pro-Exendin-4-Asp polypeptide gene in Escherichia coli

[0055] Shake the recombinant expression bacteria in liquid LB medium at 37°C with 1% inoculum size overnight, transfer 8% inoculum amount to corn steep liquor fermentation broth, add lactose at a final concentration of 5 mmol / L to induce expression after 4 hours of culture, and collect after 12 hours Bacteria, reserved samples for SDS-PAGE analysis (see attached Figure 4 ). The fusion protein reached a stable maximum expression level 12 hours after induction, and was expressed in a soluble form in the cell.

Embodiment 3

[0056] The separation and purification of embodiment 3 fusion protein

[0057] Select high-expression strains, inoculate them in LB culture, and culture overnight at 37°C; transfer the overnight culture solution into corn steep liquor medium, expand culture at 37°C, and select the optimal fermentation conditions to obtain fermentation cells. Centrifuge at 12000r / min for 15min, and save the supernatant and sediment as samples. pH 7.4, 50mmol / L Tris·HCl rinse twice to precipitate the bacteria; add the prepared bacteria lysate (4ml / g bacteria) to the collected bacteria, place it on a shaker at 37°C and shake vigorously overnight , to fully lyse the bacteria; take out the lysate when the solution is not viscous, centrifuge at 12,000r / min for 15min, collect the precipitate, weigh it, and mark the supernatant and precipitate.

[0058] The supernatant of the cell lysate is filtered with a 0.45 μm filter membrane to ensure clarification and no particles, so as not to block the column...

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Abstract

The invention belongs to the field of genetic engineering bioactive peptide and discloses an Exendin-4 analogue, namely Pro-Pro-Exendin-4-Asp peptide, a preparation method thereof and application of the Exendin-4 analogue. A fusion expression preparation technology platform in the laboratory is utilized, and therefore engineering bacteria of the Pro-Pro-Exendin-4-Asp peptide in fusion expression can be created in an Escherichia coli cell. The purpose peptide, Pro-Pro-Exendin-4-Asp, can be obtained through acid cleavage of fusion protein, and the purpose peptide is obtained through nickel affinity chromatography separation. The Pro-Pro-Exendin-4-Asp peptide can effectively reduce blood sugar after normal male institute of cancer research (ICR) mice take glucose load orally and can not cause glycopenia, and secretion of insulin can be promoted effectively. The Pro-Pro-Exendin-4-Asp peptide does not have obvious influences on mice fasting blood-glucose and is capable of greatly restraining squirm of semi-solid chyme in intestinal canals, and therefore the Pro-Pro-Exendin-4-Asp peptide has good clinical application prospect on type II diabetes.

Description

technical field [0001] The invention relates to the field of preparation of active peptides by genetic engineering, and discloses the construction of an Exendin-4 analogue Pro-Pro-Exendin-4-Asp peptide and the fusion expression engineering bacteria of the peptide, acid hydrolysis of the fusion protein, and preparation by nickel column affinity chromatography Pro-Pro-Exendin-4-Asp peptide method and in vivo animal experiments were used to detect its effects on blood glucose and serum insulin levels, fasting blood glucose and intestinal peristalsis in normal male ICR mice after oral glucose load. Background technique [0002] In 2005, the US FDA approved chemically synthesized Exendin-4 for the treatment of patients with type 2 diabetes (trade name Byta) whose blood sugar was not well controlled by metformin and sulfonylureas. As the first approved incretin analogue, Exendin-4 has received a lot of attention. Its various physiological functions are aimed at the common abnorma...

Claims

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Application Information

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IPC IPC(8): C07K14/575C12N15/16C12N15/63C12N1/21C12P21/02C07K1/22A61P3/10C12R1/19
Inventor 李泰明谷春娇李苹苹平菡樊妮刘景晶屠致力冯如杰
Owner CHINA PHARM UNIV
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