rhTSG6-FNIII-C fusion protein and application thereof in skin care composition and preparation method of the rhTSG6-FNIII-C fusion protein

A rhtsg6-fniii1-c, fusion protein technology, applied in the preparation methods of peptides, preparations for skin care, methods based on microorganisms, etc. The problem is to achieve the effect of simple preparation method, less skin allergy and low production cost

Active Publication Date: 2021-06-01
WUHU TIANMING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibiotics will affect the skin immune system, resulting in fragile and sensitive skin; fungicides will destroy the micro-ecosystem of the skin; the effect of traditional Chinese medicine products is relatively mild, but the effect is slow and the treatment cycle is long
[0006] In the market of functional cosmetics such as anti-wrinkle and anti-acne, more and more products use proteins with corresponding functions as active ingredients, but there are no products with rhTSG6-FNⅢ1-C fusion protein as active ingredients, nor There are relevant patent records

Method used

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  • rhTSG6-FNIII-C fusion protein and application thereof in skin care composition and preparation method of the rhTSG6-FNIII-C fusion protein
  • rhTSG6-FNIII-C fusion protein and application thereof in skin care composition and preparation method of the rhTSG6-FNIII-C fusion protein
  • rhTSG6-FNIII-C fusion protein and application thereof in skin care composition and preparation method of the rhTSG6-FNIII-C fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Preparation of recombinant expression vector for producing rhTSG6-FNⅢ1-C fusion protein and transformation of recombinant microorganism

[0072] The optimized gene encoding the rhTSG6-FNⅢ1-C fusion protein, the recombinant expression vector and the transformed recombinant microorganism were prepared by the following methods.

[0073] Using the gene encoding rhFNⅢ1-C protein and rhTSG-6 used as a chaperone protein as a template, an optimized rhTSG6-FNⅢ1-C fusion protein encoding 349 amino acids that can be expressed in host microorganisms was produced and synthesized The fragment of the gene (SEQ ID NO: 1), which was amplified by the primers shown in Table 1 through overlap extension PCR technology, was treated with BamHI and HindIII double enzymes, and the pET-32a vector treated with the same double enzymes connect.

[0074] Table 1 PCR amplification primers

[0075]

[0076] Thus, the pET-32a-rhTSG-6-FNIII1-C recombinant plasmid was produced. For the identificati...

Embodiment 2

[0080] Expression Induction, Isolation and Purification of rhTSG6-FNⅢ1-C Fusion Protein

[0081] Fermentation tank fermentation: Prepare 2 bottles of LB medium, inoculate after high-pressure steam sterilization, and culture overnight at 37°C in shake flasks; the next day, prepare 20L system fermenter medium and dissolve it; install the pH electrode and dissolved oxygen electrode, and add the medium , when the culture medium is heated to 37°C, add ammonia water to adjust the pH to 7.0, and 1mL of antifoaming agent to reduce the influence of foam during fermentation on the experiment; adjust the "temperature T" option to sterilization mode (121°C, 20min), while irradiating the room with ultraviolet rays to establish a relatively closed and sterile environment; when the sterilization in the tank is completed and the temperature drops to 37°C, the alcohol spray can is sprayed on the top of the tank to burn, and the feed inlet is opened in the fire ring. Pour the seed liquid into t...

Embodiment 3

[0089] Identification of rhTSG6-FNⅢ1-C fusion protein by Western blot

[0090] The purified product of rhTSG6-FNⅢ1-C fusion protein prepared in Example 2 was separated by polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a membrane by wet transfer method. After the transfer, the PVDF membrane was immersed in the blocking solution and placed at 37°C for 60 minutes to block. The blocked PVDF membrane was immersed in a solution of primary antibody (rabbit anti-human TSG-6 polyclonal antibody from Abcam, diluted 1:2000) diluted in blocking solution, and incubated overnight at 4°C. Rinse the PVDF membrane 3 times with TBST buffer on a shaker, 5 minutes each time. The washed PVDF was immersed in the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG, diluted 1:50000) diluted in blocking solution, and incubated at 37°C for 1 hour. Then soak the PVDF membrane with TBST buffer 3 times, 5 minutes each time. Freshly prepared ECL working solution: E...

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Abstract

The invention relates to the technical field of cosmetics, and discloses an rhTSG6-FNIII-C fusion protein, an application of the rhTSG6-FNIII-C fusion protein in a skin care composition and a preparation method of the rhTSG6-FNIII-C fusion protein. The rhTSG6-FNIII-C fusion protein is prepared through a genetic engineering prokaryotic expression technology. The specific method comprises the following steps: carrying out codon optimization on a gene sequence of SEQ ID NO: 1; constructing a recombinant expression vector and transforming; inducing and expressing the recombinant Escherichia coli; and separating and purifying an expression product, so that the rhTSG6-FNIII-C fusion protein is prepared. The skin care composition has various dosage forms, such as an aqueous solution, an ointment, a suppository, a cream and a mask, and has good skin care effects of improving skin wrinkles, keeping skin elasticity, reducing skin inflammation and allergy risks and the like, so that the skin care composition can be applied to the field of functional cosmetics or medical cosmetology.

Description

technical field [0001] The invention relates to a recombinant human tumor necrosis factor α-stimulated gene-6-fibronectin Ⅲ 1-C (recombination human tumor necrosis factor α-stimulated gene-6-fibronectin Ⅲ 1-C, rhTSG6-FN Ⅲ 1) capable of enhancing skin cell proliferation and skin tissue repair -C) a fusion protein, and a skin care composition for improving skin wrinkles, maintaining skin elasticity, and reducing skin inflammation and allergies. It contains the same active ingredients. Background technique [0002] Human skin is composed of epidermis, dermis, subcutaneous tissue and skin appendages, and has functions such as protection, temperature regulation, metabolism and sensation. The state of the epidermis and dermis is closely related to the state of the skin, and the homeostasis of the cellular environment composed of cells and extracellular matrix is ​​related to the state of the skin. [0003] Fibronectin (FN) is a high molecular weight (about 440kDa) dimeric glycop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12P21/02C07K1/22A61K8/64A61Q19/08A61Q19/00A61Q19/02C12R1/19
CPCC07K14/78C07K14/4718C12N15/70A61K8/64A61Q19/08A61Q19/00A61Q19/02C07K2319/00C12N2800/22
Inventor 王明丽何志远蒋敏之吴博周炜夏兵兵赵俊张俊玲余莉严德祥
Owner WUHU TIANMING BIOTECH CO LTD
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