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Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof

A technology of human serum albumin and serum albumin, which is applied in the direction of peptide/protein components, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of low efficiency and high production cost, extend the half-life and reduce the burden of treatment , Improve the effect of bioavailability and therapeutic value

Inactive Publication Date: 2012-12-12
无锡和邦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production cost of chemically coupling Exendin-4 with HSA in vitro is high and the efficiency is low

Method used

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  • Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof
  • Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof
  • Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: human albumin gene cloning

[0054] The human HSA mature peptide gene was prepared by RT-PCR, and the primers were designed as follows:

[0055] HSA1:5'-GATGCACACAAGAGTGAGGTGCTCA-3',

[0056] HSA2: 5'-AAGCTAGCTT ATAAGCCTAA GGCAGCTTG ACTTGC-3'.

[0057] Human fetal liver tissue was taken, and total RNA was prepared by the Trizol method. RT-PCR amplification: In a 0.2mL PCR tube, add 25μL of 2×RT PCR buffer, 3μL of total RNA (50ng), primers HSA1 0.5μL (25μM), HSA2 0.5μL (25μM), RT-Phusion mixed enzyme 1 μL, add DEPC water to a final volume of 50 μL. PCR conditions: 45°C for 30min; then 94°C for 2min; then 94°C for 30sec; 55°C for 1min, 72°C for 2min, a total of 30 cycles. Finally, keep warm at 72°C for 5 minutes. The PCR product was subjected to agarose gel electrophoresis to observe the amplification result and product size. Then use the gel recovery method to purify the HSA gene fragment. The specific method is to cut off the DNA band with a molecula...

Embodiment 2

[0058] Embodiment 2: Exendin-4 gene synthesis

[0059] The Exendin-4 gene is prepared by artificial synthesis. The sequence according to SEQ ID NO: 5 was synthesized from the Exendin-4 gene of Shanghai Sangon Bioengineering Technology Service Co., Ltd., and a StuI restriction site was provided at the 5' end.

Embodiment 3

[0060] Embodiment 3: Construction and cloning of Exendin-4-HSA fusion gene

[0061] In this experiment, the Exendin-4 and HSA genes were directly fused to form the Exendin-4-HSA fusion gene by overlapping PCR method as an example to illustrate the method of synthesizing the fusion gene. The specific method is as follows: without adding primers and polymerase, mix the components according to the following system.

[0062] 5×phusion HF buffer 20 μL, dNTPs (10 mM) 2 μL, Exendin-4 DNA fragment 2 μL, HSA DNA fragment 2 μL, ddH 2 O 69 μL for a total volume of 95 μL.

[0063] Denature at 99°C for 10 min, then cool naturally at room temperature. Add 0.5 μL of Phusion high-fidelity enzyme, incubate at 72°C for 15 minutes, and then add primers

[0064] Ex1: (5′-TA AGGCCT CACGGTGAAGGTACTTTCACTT-3′,Stu1) (50 μM) and

[0065] HSA2: (5′-AAGCTAGCTTATAAGCCTAAGGCAGCTTGACTTGC-3′, Nhe1) (50 μM) 2 μL each, Phusion high-fidelity enzyme 0.5 μL, for conventional PCR amplification:

[0066] Pr...

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Abstract

The invention discloses a fusion protein of exendin-4 peptide and human serum albumin (HSA) and a preparation method thereof, belonging to the technical field of long-term recombination fusion protein medicine. The invention relates to a preparation method and a product of fusion protein of insulin secretion promoting peptide (Exendin-4) and human serum albumin (HSA). The fusion protein is characterized in that a genetic engineering method is adopted to directly fuse a single Exndin-4 peptide and HAS and without connecting peptide at the middle part to form stable and long half-life period Exendin-4-HAS fusion protein, and is applied in a treatment of non-insulin-dependent diabetes and obesity.

Description

technical field [0001] A fusion protein of Exendin-4 peptide and human serum albumin (HSA) and a preparation method thereof belong to the technical field of long-acting recombinant fusion protein drugs. [0002] In the present invention, by directly fusing a single Exendin-4 polypeptide with human serum albumin, the half-life of Exendin-4 in the body can be extended while maintaining the efficacy of Exendin-4, improving bioavailability and therapeutic value, and reducing the burden of treatment on patients , has a good application prospect in the field of pharmacy. The invention relates to the preparation of long-acting medicine for treating non-insulin-dependent type II diabetes. Background technique [0003] Diabetes is a chronic disease that seriously threatens the health of people all over the world. The number of people with diabetes is increasing. By 2030, there will be 435 million people with diabetes worldwide. The incidence of diabetes in my country is as high as ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/81C12P21/02A61K38/38A61K38/22A61P3/10A61P3/04C12R1/865
Inventor 杨建良余传信孟如杰冯炜
Owner 无锡和邦生物科技有限公司
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