Saccharomyces cerevisiae expressed long-acting recombinant fibronectin and application thereof in cosmetics

A technology for fibronectin and Saccharomyces cerevisiae, applied in the emerging biomedical field, can solve the problems of low feasibility of full sequence expression, large molecular weight of natural FN, and low biological activity of FN protein.

Inactive Publication Date: 2021-07-30
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the complex process and high cost of natural purification of FN; the large molecular weight of natural FN and the low feasibility of full sequence expression limit its popularization and application; existing prokaryotic expression systems are prone to formation of inclusion bodies, and the obtained FN protein has low biological activity To solve the techn

Method used

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  • Saccharomyces cerevisiae expressed long-acting recombinant fibronectin and application thereof in cosmetics
  • Saccharomyces cerevisiae expressed long-acting recombinant fibronectin and application thereof in cosmetics
  • Saccharomyces cerevisiae expressed long-acting recombinant fibronectin and application thereof in cosmetics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] This example discloses a long-acting recombinant fibronectin expressed by Saccharomyces cerevisiae. It overcomes the complexity and high cost of the natural purification process of FN; the large molecular weight of natural FN and the low feasibility of full sequence expression limit its popularization and application; the existing prokaryotic expression system is easy to form inclusion bodies, and the obtained FN protein has low biological activity and other defects.

[0063] The long-acting recombinant fibronectin is rFNIII1C-HSS. The nucleotide sequence of the long-acting recombinant fibronectin is shown in Seq ID NO.1, and the amino acid sequence corresponding to the nucleotide sequence of the long-acting recombinant fibronectin is shown in Seq ID NO.2. The nucleotide sequence of long-acting recombinant fibronectin includes the functional fragment of FN, HAS; the functional fragment of FN includes the FNIII1C unit; the FNIII1C unit is a functional fragment of the C-...

Embodiment 2

[0109] This embodiment discloses a method for detecting the adhesion-promoting activity of bovine kidney cells expressed by Saccharomyces cerevisiae and expressing long-acting recombinant fibronectin. Used to verify the attachment activity of the long-acting recombinant fibronectin FNIII1C-HSS promoting bovine kidney cells (MDBK) as in Example 1. The detection method comprises reagent preparation and cell growth promoting activity determination.

[0110] Reagents include: complete culture solution, serum-free culture solution, digestion solution and PBS buffer.

[0111] The preparation of each cultivation and reagent in the present embodiment specifically includes:

[0112] (1) Complete culture medium: Measure 10ml of fetal bovine serum, 1ml of double antibody, and add 90ml of DMEM culture medium.

[0113] (2) Serum-free culture medium: Measure 1ml of double antibody, add 99ml of 1640 culture medium.

[0114] (3) Digestive fluid: 0.25% trypsin.

[0115] (4) PBS buffer solu...

Embodiment 3

[0124] This example provides an experiment of using rFNIII1C-HSA in repairing skin damage, which is used to verify the effect of rFNIII1C-HSA in repairing skin damage in Example 1. Experiments on mouse models have proved that FNIII1C-HSA can effectively promote skin wound repair and significantly increase the healing rate of injuries. The implementation includes the following steps:

[0125] (1) Test groups

[0126] 4-5 week-old experimental rats were divided into 2 groups, 10 in each group, with a body weight of about 250 g, and marked with ear marks.

[0127] (2) Test treatment

[0128] A punch was used to punch a hole (0.5 cm in diameter) in the back, and a PBS smearing control group and a rFNIII1C-HSS (1 mg / ml) smearing experimental group were respectively set up, and smeared 3 times a day until the wound healed. The two groups of rats were raised in a warm and dry environment, and pictures were taken every day to observe and record the wound healing.

[0129] (3) Expe...

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Abstract

The invention discloses a saccharomyces cerevisiae expressed long-acting recombinant fibronectin and an application of the long-acting recombinant fibronectin in cosmetics. The nucleotide sequence of the long-acting recombinant fibronectin is shown as Seq ID NO.1, and the long-acting recombinant fibronectin comprises a functional fragment of FN and HAS; the functional fragment of the FN comprises an FNIII1C unit; and the FNIII1C unit is a functional fragment at the C end of the first III-type FN repetitive unit in the FN. FNIII1C is a functional fragment at the C end of a first III type FN repetitive unit in natural human FN, and has the functions of resisting tumors, resisting metastasis and inhibiting angiogenesis. The protein can also enable FN in a living body to be subjected to super-polymerization, and the cell adhesion promoting and cell migration resisting capabilities of the FN are greatly enhanced. The fusion HAS effectively improves the stability of the recombinant protein. The long-acting recombinant fibronectin disclosed by the invention can be used for improving the adherence rate and convergence rate of cells, shortening the cell convergence time, enabling the morphological structure of the cells to be good, enhancing the metabolic rate and remarkably improving the protein synthesis speed, and has the effect of promoting cell repair.

Description

technical field [0001] The present invention relates to the emerging field of biomedicine, in particular to a long-acting recombinant fibronectin expressed by Saccharomyces cerevisiae, a detection method for detecting the adhesion-promoting activity of bovine kidney cells by long-acting recombinant fibronectin, and a long-acting recombinant fibronectin expressed by Saccharomyces cerevisiae Application of recombinant fibronectin in cosmetics. Background technique [0002] Natural fibronectin (fibronectin, FN) is a high molecular weight glycoprotein and one of the most important components of the extracellular matrix, widely present in blood, body fluids and various tissues. In vertebrates, FN is highly conserved and has the same organizational structure. FN exists in the form of a dimer, which is composed of two polypeptide chains with a molecular weight of about 250 kDa connected by a disulfide bond at the C-terminus. Each chain contains a series of repeat units: 12 type I...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C07K14/78C12Q1/02A61K8/64A61Q19/00C12R1/865
CPCA61K8/64A61Q19/00C07K14/78C12N15/81G01N33/5008
Inventor 赵俊刘洁夏兵兵丁爽周炜凡玉芳李媛媛
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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