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Method for improving bioactivity of exendin fusion protein

A fusion protein and insulin-stimulating technology, which is applied in the field of biomedicine, can solve problems such as prolongation, and achieve the effect of prolonging half-life, good application prospects, and high biological activity

Active Publication Date: 2010-11-03
无锡和邦生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows creating fusion proteins that can be used with different drugs or other substances like Insulinotropic polypeptide (Incretis), Growth factor 1(IGFI) hormone, Cytokine Tumor Nerve Factor (TNF). These fusion molecules are designed specifically targeted towards certain diseases such as Type I Diabetes Mellitis and Obesity due their ability to stimulate endocrine cells called beta adrenergic receptors on pancreatic nerves, resulting in improved blood glucose levels over time compared to previous methods involving injection into patients' bodies.

Problems solved by technology

The technical problem addressed in this patented text relates to improving the effectiveness and safety of treatments against metabolism related issues associated with aging: Overall, finding ways to extend lifelong healthy lifestyles without causing any negative consequences remains challenging despite advancements made during recent years.

Method used

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  • Method for improving bioactivity of exendin fusion protein
  • Method for improving bioactivity of exendin fusion protein
  • Method for improving bioactivity of exendin fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: human serum albumin HSA gene cloning, obtaining HSA gene

[0054] Human serum albumin HSA gene can be obtained by RT-PCR. The HSA gene was amplified by PCR using human embryonic cDNA as a template. PCR amplification primers HSA primer 1 and HSA primer 2 were designed according to the HSA gene sequence SEQ ID NO:5. Its sequence is as follows:

[0055] HSA Primer 1: 5'-GATGCACACAAGAGTGAGGTTGCT-3'

[0056] HSA Primer 2: 5'- GCGGCCGC TTATAAGCCTAAGGCAGCTT-3'.

[0057] The underlined part is the NotI restriction site.

[0058] The PCR reaction system is: human embryo cDNA: 1 μL; HSA primer 1: 50 μM, 1 μL; HSA primer 2: 50 μM, 1 μL; 10×pfu polymerase buffer 5 μL; MgCl 2 : 25mM, 5μL; dNTP: 2mM, 4μL; pfu polymerase 1μL; ddH 2 O: Supplement to a total reaction system of 50 μL.

[0059] The PCR reaction conditions were: 94°C, 2min; then, 94°C, 30sec; 55°C, 30sec; 72°C, 90sec, for a total of 30 cycles. The PCR product was subjected to gel electrophoresis wit...

Embodiment 2

[0060] Embodiment 2: Exendin-4-GLP-1 gene synthesis

[0061] According to the requirement of SEQ ID NO: 6, the gene sequence of Exendin-4-GLP-1 was synthesized by Shanghai Handsome Biotechnology Co., Ltd., and a 5' end sequence of HSA gene was added to its 3' end.

Embodiment 3

[0062] Example 3: Exendin-4-GLP-1 and HSA gene fusion

[0063] In this example, the fusion gene of Exendin-4-GLP-1 and HSA gene is taken as an example to illustrate the cloning method of the fusion gene. This method is also suitable for the cloning of fusion genes of HSA gene and other genes.

[0064]According to SEQ ID NO: 6, design and synthesize a primer:

[0065] EXT 1: 5′-TT TACGTA CACGGTGA AGGTACTTTCACT-3′

[0066] The underlined part is the SnaB1 restriction site.

[0067] The fusion protein gene was prepared using the Overlap PCR reaction method. details as follows.

[0068] The reaction system for Overlap PCR is: Exendin-4-GLP-1 DNA: 2 μL; HSA DNA: 2 μL; 10×pfu polymerase buffer 5 μL; 25 mM MgCl 2 5 μL; 4 μL of 2 mM dNTP; 1 μL of pfu polymerase; ddH 2 O was made up to a total reaction volume of 50 μL.

[0069] The PCR conditions are: start, 94°C, 2min; then, 94°C, 30sec; 55°C, 30sec; 72°C, 2min; a total of 5 cycles. Then, add 50 μM primer EXT1: 2 μL and 50 μ...

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Abstract

The invention discloses a method for improving the bioactivity of an exendin fusion protein, which belongs to the technical field of biopharmaceuticals. In the method, an Exendin-4 and GLP-1 are connected in series first and then are connected with human serum albumin HSA or human immunoglobulin Fc to form the fusion protein, so as to increase the bioactivity of the exendin fusion protein. The fusion protein is expressed as: (Exendin-4-GLP-1)n-HSA, (GLP-1-Exendin-4)n-HSA, (Exendin-4-GLP-1)n-Fc or (GLP-1-Exendin-1)n-Fc, wherein the value of n ranges from 1 to 5. The fusion protein prepared by the method has higher bioactivity than a fusion protein Exendin-4-HSA or a GLP-1-HSA protein. The fusion protein of the invention can be used for preparing medicaments for treating non-insulin-dependent diabetes, various symptoms of the non-insulin-dependent diabetes and obesity.

Description

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Claims

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Application Information

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Owner 无锡和邦生物科技有限公司
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