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A kind of gene recombinant collagen oligopeptide mys-1 and its preparation method and application

A technology of MYS-1 and collagen oligopeptide, applied in the field of genetic engineering, can solve the problem of high cost of collagen, achieve better biological safety, small molecular weight, and broad application prospects

Active Publication Date: 2021-12-28
广州暨大美塑生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional extraction technology obtains a series of hydrolysates by hydrolyzing natural collagen, but the application risk of immunogenicity is unavoidable; the cost of chemically synthesizing collagen is high; genetic recombination technology has high yield, low cost, and the production of protein, polypeptide or oligopeptide The advantages of high activity and no hidden dangers of viruses make the recombinant small molecule collagen peptide have good industrialization prospects, especially the recombinant collagen oligopeptide with high stability, which has huge market application potential

Method used

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  • A kind of gene recombinant collagen oligopeptide mys-1 and its preparation method and application
  • A kind of gene recombinant collagen oligopeptide mys-1 and its preparation method and application
  • A kind of gene recombinant collagen oligopeptide mys-1 and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Construction and Expression of Expression Engineering Bacteria pET32a-MYS-1 / BL21(DE3)

[0061] Specific steps are as follows:

[0062] (1) Synthesis of MYS-1 gene:

[0063] Primer design:

[0064] Primer 1:

[0065]

[0066] Primer 2:

[0067]

[0068] GGTACC is the KpnI restriction site, CTCGAG It is the XhoI restriction site.

[0069] The amount of each component of annealing: (primer concentration is 1OD dissolved in 400μL ddH 2 O, reaction system: 20μL)

[0070] Primer 1: 2 μL, Primer 2: 2 μL, T4 PNK 1 μL (10 U), ATP 20 mM, ddH 2 O replenish water to 20 μL;

[0071] The procedure for synthesizing the MYS-1 gene: 95°C, 5min; 95°C, 1min, 56°C, 1min, 72°C, 1min, 30 cycles; 72°C, 10min;

[0072] Cool naturally to room temperature to obtain the product for later use.

[0073] (2) Construction of recombinant vector pET32a-MYS-1:

[0074] Use KpnI enzyme and XhoI enzyme to perform double enzyme digestion on the plasmid pET32a (purchased from Shanghai Sa...

Embodiment 2

[0080] Purification, preparation and identification of recombinant human collagen oligopeptide MYS-1

[0081] Pipette and mix 2mL of medium into the column (Ni-NTA column, GenScript Biotechnology Co., Ltd., 10mL column volume), add 4 times the column volume of Lysis equilibration buffer to equilibrate the chromatography medium; The supernatant was loaded onto the column at a flow rate of 1.0mL / min; with 8 times the column volume of washing buffer (50mM Na 2 HPO 4 , 0.3M NaCl, 10~50mM imidazole, pH=8.0) to pass through the column at 1.0mL / min to wash away the impurity protein or fusion protein not bound by affinity, and use 10 times column volume of elution buffer at 1.0mL / min Min passed through the column, collected the eluate, took the sample effluent, washing effluent, and eluted effluent for SDS-PAGE electrophoresis to detect the purification effect of the fusion protein Trx-6His-MYS-1. The fusion protein with most of the foreign proteins removed was obtained in the solut...

Embodiment 3

[0090] Stability detection of recombinant human collagen oligopeptide MYS-1

[0091] Recombinant human collagen oligopeptide MYS-1 was dissolved in 20mM sodium phosphate buffer (containing 150mM sodium chloride) (pH 8.0) at a concentration of 1mg / mL, incubated at 37°C, and analyzed using a liquid chromatography-mass spectrometry system (LC-MS) detects the stock flow of recombinant human collagen oligopeptide MYS-1 in the solution over time (1 to 6 weeks), calculates the depletion rate of MYS-1 over time, and uses marine collagen peptide (MCP) and recombinant collagen Peptide MJLGG-34 was used as a control. The genetic recombinant collagen-like peptide MJLGG-34 is disclosed in "201811505180.3, Genetic recombinant collagen-like peptide MJLGG-34 and its preparation method and application".

[0092] Such as figure 2As shown, when incubated for 1 week, MCP was reduced by 22.1%, and when incubated for 4 weeks, MCP was reduced by 66.7%; 18.9% and 24.0%; while the gene recombinant...

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Abstract

The invention discloses a gene recombinant collagen oligopeptide MYS‑1, a preparation method and application thereof, and belongs to the technical field of genetic engineering. Collagen oligopeptide MYS-1 obtained through gene recombination, compared with natural collagen or collagen-like peptide, has small molecular weight, easy preparation and high efficiency, and has higher stability, better transdermal absorption rate and higher Biological activity, good biocompatibility and biosafety, good anti-oxidation, anti-aging, promoting repair, inhibiting melanin production and other biological activities, can significantly promote skin oxidative damage repair, reduce melanin production and resist aging , and has no obvious toxic and side effects; it can be used in food, health products, cosmetics, etc., with a wide range of applications, and has good industrial application value and prospects. The preparation method of the invention is safe and efficient, has high production efficiency, low production cost, high stability of the produced oligopeptide, and broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a gene recombinant human collagen oligopeptide MYS-1 and its preparation method and application. Background technique [0002] Collagen is a kind of structural protein in the animal body, accounting for 25% to 35% of the total protein content of the human body. It is the main component of the extracellular matrix and plays an important role in maintaining the normal physiological functions of cells, tissues and organs and repairing damage. Collagen has extremely low immunogenicity, degradability, biocompatibility, bioabsorption, mechanical, hemostatic, non-toxic and cell growth promotion functions. It is widely used in clinical medicine, biomaterials, biomedicine, food chemical industry and Cosmetics industry research has a wide range of applications. Studies have shown that collagen is a key functional factor that regulates skin water content and PH value, regulate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/70A23L33/18A61K8/65A61Q19/00A61Q19/02A61Q19/08
CPCC07K14/78C12N15/70A23L33/18A61K8/65A61Q19/00A61Q19/02A61Q19/08A23V2002/00A23V2200/30A23V2200/302A23V2200/318A23V2250/55
Inventor 马义万阳徐晶夏商周
Owner 广州暨大美塑生物科技有限公司
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