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Recombinant short peptide, production method and application thereof, and application of recombinant short peptide in promoting wound healing

A production method and short peptide technology, applied in the production method and application, and the field of recombinant short peptides, to achieve the effect of solving delayed wound healing and non-healing and good stability

Inactive Publication Date: 2020-04-28
河北柯瑞生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And how to make the growth factor maintain its biological activity in a certain period of time has not been reported so far

Method used

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  • Recombinant short peptide, production method and application thereof, and application of recombinant short peptide in promoting wound healing
  • Recombinant short peptide, production method and application thereof, and application of recombinant short peptide in promoting wound healing
  • Recombinant short peptide, production method and application thereof, and application of recombinant short peptide in promoting wound healing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Screening and Synthesis of Genes

[0029] Firstly, codon optimization was carried out on the gene sequence of rEGF, and at the same time, Escherichia coli preferred stop codon TAA was added to the 3' end, and the gene sequence as shown in SEQ ID NO:1 was designed, specifically as follows:

[0030] ATTAGCACAAAATAATGGGAATAAAGGGAACAGTTCTGAACCAAACTGCTGACATTGGGCTCCTGTCCTGGCTCTCCTGTAAAGGAAATTACATGGATGAGGAGGTCCAAGTCTCTGTCACCAGGGAGCACTCACTGGAATGGGTGAGCAGAACCCTAGGGTATTAGAAGGCAGGACTGAGTTCTAATTTTCCTGGGTCCCCAGCACCTGAAACTGTCAAGTTCAT.

[0031] The length of the nucleic acid is 237bp, and a third-party company is commissioned to synthesize the DNA sequence of the gene.

Embodiment 2

[0032] Example 2 Construction and Screening of Escherichia coli Transformants

[0033] The synthesized vector pUC-rECF containing the gene sequence of SEQ ID NO: 1 was digested with restriction endonucleases Nde I and Xho I at 37°C for 1 h, and the small fragments after digestion were recovered on agarose gel to obtain the 240bp target fragment, see figure 1 . Ligate with the expression vector pET-24b that has been cut with the same double enzymes under the action of T-DNA ligase at 16°C for 1 hour, and transform the ligated product into competent Escherichia coli BL21 by calcium chloride method. Cultivate in culture medium and select positive bacteria.

[0034] The construction of the pET-24b-rEGF recombinant plasmid, the connection system is as follows:

[0035] pET-24b digested empty vector 1 μL

[0036] 2.5 μL of target gene after enzyme digestion

[0037] T4 DNA Ligase 1μL

[0038] T4 DNA Ligase Buffer 2.5μL

[0039] Add dd H 2 0 to 25 μL.

[0040] For the specif...

Embodiment 3

[0042] Example 3 induced expression

[0043] Inoculate the positive strains in the LB liquid medium containing kanamycin, shake at 200 r / min at 37°C for overnight activation, and re-inoculate the LB medium at a ratio of 1:100 to detect the growth density of the bacteria. D. 600 When it reached 0.4, IPTG was added to make the final concentration 0.3 mmol / L, and the expression was induced at 37°C for 7 hours, and the precipitate was collected by centrifugation to obtain the bacteria.

[0044] Protein expression detection: take the bacteria collected in step (3), add PBS buffer and mix well, break up by ultrasonic in an ice bath, add 5× protein loading buffer to the expression bacteria solution, boil and take 20uL for 15% SDS-PAGE (recipe See Table 1) Detection. Analyze the expression level of the target protein in the prokaryotic system, as shown in the attached figure 2 As shown, compared with the control bacteria (the Escherichia coli strain containing the blank expression...

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Abstract

The invention relates to a recombinant short peptide, and a production method and application thereof. The coding gene of the recombinant short peptide is shown as SEQ ID NO:1, and the recombinant short peptide is obtained by performing codon optimization on an rEGF nucleotide sequence and adding an escherichia coli preference stop codon TAA at the 3' end. The recombinant short peptide is obtainedby inserting the SEQ ID NO:1 between enzyme cutting sites NdeI and XhoI of an expression vector pET-24b to obtain a recombinant expression plasmid, converting the recombinant expression plasmid intoa competent escherichia coli BL21 by a calcium chloride method to obtain a positive bacterium, performing induction expression or fermentation culture, and performing separating and purifying. The recombinant short peptide has good stability at room temperature, and has great advantages in solving symptoms of delayed healing, nonhealing and the like of a wound surface.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant short peptide, its production method and application. Background technique [0002] Wound is the damage of normal skin (tissue) caused by external injury factors such as surgery, external force, heat, electric current, chemical substances, low temperature and internal factors such as local blood supply disturbance, etc., often accompanied by skin integrity destruction and loss of a certain amount of normal tissue. [0003] Wound healing starts when the normal function and integrity of the skin is disrupted. Many factors, such as population aging, malnutrition, stress response, bacterial infection, and diabetes mellitus, affect wound healing, leading to delayed or non-healing of various acute and chronic wounds. At present, delayed wound healing and non-healing are still a major medical problem, and new treatment options are urgently needed. [0004] In th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12P21/02C07K14/485C07K1/22A61K38/18A61P17/02
CPCC07K14/485C12N15/70A61K38/1808A61P17/02C12N2800/22
Inventor 左明星夏中博齐磊李杰孙敬超刘鑫
Owner 河北柯瑞生物医药有限公司
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