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Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof

A technology of epidermal growth factor and metallothionein, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve problems such as poor results, reduce the frequency of medication, reduce the amount of medication, and stabilize sex increasing effect

Inactive Publication Date: 2010-11-03
SHANGHAI SIRUIBAO BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to provide a kind of fusion protein of human epidermal growth factor and metallothionein, the fusion protein of described this human epidermal growth factor and metallothionein will solve the independent human epidermal growth factor or metallothionein in the prior art. Technical problem that metallothionein is ineffective in the process of treating burns, or scalds, or wounds, or ulcers

Method used

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  • Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof
  • Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof
  • Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof

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Embodiment 1

[0040] Synthesis and assembly of embodiment 1 human epidermal growth factor gene

[0041] The entire sequence of the novel human epidermal growth factor fusion protein is artificially designed according to the customary codons of Escherichia coli, and is divided into six oligonucleotides for synthesis (respectively named as EU1, EU2, EU3, ED1, ED2, ED3) (such as figure 1 shown), the base sequence of oligonucleotide EU1 is shown in SEQ ID No: 5, the base sequence of oligonucleotide EU2 is shown in SEQ ID No: 6, and the base sequence of oligonucleotide EU3 is shown in Shown in SEQ ID No: 7, the base sequence of oligonucleotide ED1 is shown in SEQ ID No: 8, the base sequence of oligonucleotide ED2 is shown in SEQ ID No: 9, the base sequence of oligonucleotide ED3 is shown in The base sequence is shown as SEQ ID No: 10, which was synthesized by Shanghai Yingjun Biotechnology Co., Ltd., in which EU1 and ED1 two oligonucleotide chains form a sticky end of EcoR I after annealing, whi...

Embodiment 2

[0043] Example 2 Synthesis and assembly of human metallothionein gene

[0044] According to the human metallothionein (MT1M) sequence (as shown in SEQ ID No: 4) that has been published, its coding gene (as shown in SEQ ID No: 11) is designed using Escherichia coli customary codons, and in the encoding gene An XbaI site was designed at the 5' end, and a Hind III site was designed at its 3' end for cloning. The human metallothionein coding gene entrusted Shanghai Yingjun Biotechnology Company to carry out the whole gene synthesis.

[0045] Shanghai Yingjun Biotechnology Co., Ltd. provided the cloning plasmid with the above-mentioned synthetic human metallothionein coding gene verified by sequencing, and named it pGM01.

Embodiment 3

[0046] The construction of embodiment 3 fusion protein Escherichia coli expression vector

[0047] Inoculate the Escherichia coli strain containing the recombinant plasmid named pGE04, the Escherichia coli strain containing the recombinant plasmid named pGM01, and the Escherichia coli strain containing the Escherichia coli expression vector pBV220 into 10ml LB medium containing ampicillin 50mg / L Medium, 37°C shaking culture overnight, the next day according to Qiagen company plasmid extraction kit manual instructions, respectively, to extract the plasmid. The recombinant plasmid pGE04 was digested with EcoR I and Xba I, the recombinant plasmid containing pGM01 was digested with Xba I and Hind III, and the expression vector pBV220 was digested with EcoR I and Hind III. Specific conditions: 10 μl reaction system, in the system Add 2 μl of plasmid, use 10 activity units of each restriction enzyme (NewEngland Biolabs Company), add 1 μl of 10× buffer, make up with deionized water, ...

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Abstract

The invention provides a fusion protein of a human epidermal growth factor and metallothionein, which comprises a first part and a second part, wherein the first part is the human epidermal growth factor, the second part is the human metallothionein, and the human metallothionein is positioned at the C terminal of the human epidermal growth factor. The invention further provides an application of the fusion protein in the preparation of drugs for treating burns or scalds or wounds or ulcers and the application of the fusion protein in the preparation of skin nursing drugs. Compared with the original human epidermal growth factor and the metallothionein, the fusion protein can increase the stability, reduce the clearance rate, reduce the degradation, bring benefits and convenience to the use of the EGF-MT fusion protein and further reduce the using dosage and the medication frequency. Simultaneously, the formed fusion protein is a bifunctional molecule which has double effects of EGF and MT.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and relates to a fusion protein, in particular to a fusion protein of human epidermal growth factor and metallothionein, a preparation method and application thereof. Background technique: [0002] In 1962, Cohen discovered epidermal growth factor (EGF) from the submandibular gland of mice, and determined the amino acid composition of EGF in 1975, while the human EGF gene was only cloned in 1986. Natural epidermal growth factor is a protein of 53 amino acids, with a molecular weight of about 6000 Daltons, containing three pairs of disulfide bonds (Cohen et al., J.Biol.Chem, 1962, 237:1555-1562), and has various biological activities (Karnes et al., Epidermal growth factor and transforming growth factor alpha, 1994, Raven Press, New York). Due to the small size of EGF, it is difficult to express it in E. coli alone, but there are many data on expressing human EGF in yeast and mammalian cells. Ma Qi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/19C12N1/21C12N5/10C12P21/02A61K38/18A61P17/00A61P17/02C12R1/19C12R1/84
Inventor 孙家驹周银华
Owner SHANGHAI SIRUIBAO BIOTECH
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