Saccharomyces cerevisiae expressed long-acting recombinant III-type collagen and application thereof in cosmetics

Abstract

The invention discloses a long-acting recombinant III-type collagen expressed by saccharomyces cerevisiae and an application of the long-acting recombinant III-type collagen in cosmetics. The amino acid sequence of the long-acting recombinant III-type collagen comprises a basic repetitive unit and a terminal amino acid sequence. The basic repetitive unit comprises a human type III collagen peptide fragment, and the amino acid sequence of the human type III collagen peptide fragment is GERGAPGFRGPAGPNGIPGEKGPAGERGAP. The terminal amino acid sequence comprises a human type II collagen peptide fragment, and the amino acid sequence of the human type II collagen peptide fragment is GPPGPCCGGG. The long-acting recombinant III-type collagen provided by the invention has good biocompatibility and good cell adhesion, can promote formation of new cells, has a hemostatic function, and has a stronger function of promoting growth of epithelial cells. Compared with collagen extracted from animal tissues and foreign bodies, the long-acting recombinant III-type collagen has the advantages of solubility, no virus hidden danger and low rejection reaction.

Description

technical field

[0001] The present invention relates to the emerging field of biomedicine, in particular to a long-acting recombinant type III collagen expressed by S. Application in cosmetics. Background technique

[0002] Collagen (collagen), the early definition is "the product of forming glue". The morphology of collagen is generally white, transparent, unbranched fibrils. The amino acid composition of collagen has the following characteristics: glycine (Gly, G) accounts for 33% of the total amino acid residues, that is, there is a glycine every two other amino acids (X, Y), so its peptide chain can use (G-X-Y)- to represent; X and Y can be any amino acid, but X is usually proline (Pro, P), Y is usually hydroxyproline (HyP, P * ) and hydroxylysine (Hyl, K * ). At present, more than 20 kinds of natural collagens have been isolated, which are called type I and type II collagen according to the order of discovery, and are distributed in animal skin, bones, ligaments, v...

Examples

Embodiment 1

[0033] This embodiment provides a long-acting recombinant type III collagen expressed by Saccharomyces cerevisiae, the amino acid sequence of the collagen includes basic repeating units and terminal amino acid sequences; the basic repeating units include human type III collagen peptides, human type III collagen The amino acid sequence of the peptide is GERGAPGFRGPAGPNGIPGEKGPAGERGAP; the terminal amino acid sequence includes the human type II collagen peptide, and the amino acid sequence of the human type II collagen peptide is GPPGPCCGGG.

[0034] The nucleotide sequence of the long-acting recombinant type III collagen includes a Not I restriction site, an Xba I restriction site, a start codon, a stop codon and a 6×His tag sequence. The nucleotide sequence of the Not I restriction site is GCGGCCGC; the nucleotide sequence of the Xba I restriction site is TCTAGA; the nucleotide sequence of the start codon is ATG; the nucleotide sequence of the stop codon is TAA .

[0035] The...

Embodiment 2

[0043] This example provides a method for detecting the activity of the long-acting recombinant type III collagen, which is used to verify the cell proliferation-promoting activity of the long-acting recombinant type III collagen described in Example 1. The cell line used in this detection method is BALB / C 3T3 cells.

[0044] The activity detection method includes reagent preparation, assay operation and assay method. Reagents include complete culture solution, maintenance culture solution, digestion solution, thiazolium blue solution, and PBS buffer. Determination operations include the preparation of recombinant human collagen test samples.

[0045] The assay method comprises the following steps:

[0046] Step 1, the cultivation of the cell line, the cell line is BALB / C 3T3 cell line;

[0047] Step 2, preparing a cell suspension, inoculating the cell suspension on a 96-well plate;

[0048] Step 3, maintaining cell survival with maintenance medium;

[0049] Step 4, addin...

Embodiment 3

[0070] This example provides an application of Saccharomyces cerevisiae expressing long-acting recombinant type III collagen in cosmetics, which uses the long-acting recombinant type III collagen as described in Example 1. The long-acting recombinant type III collagen has good biocompatibility, good cell adhesion, can promote the formation of new cells, and has hemostatic function. And as described in Example 2, the long-acting recombinant type III collagen in Example 1 has a stronger function of promoting the growth of epithelial cells. It can be seen that the long-acting recombinant type III collagen described in Example 1 has broad application prospects in cosmetics.

technical field

[0001] The present invention relates to the emerging field of biomedicine, in particular to a long-acting recombinant type III collagen expressed by S. Application in cosmetics. Background technique

[0002] Collagen (collagen), the early definition is "the product of forming glue". The morphology of collagen is generally white, transparent, unbranched fibrils. The amino acid composition of collagen has the following characteristics: glycine (Gly, G) accounts for 33% of the total amino acid residues, that is, there is a glycine every two other amino acids (X, Y), so its peptide chain can use (G-X-Y)- to represent; X and Y can be any amino acid, but X is usually proline (Pro, P), Y is usually hydroxyproline (HyP, P * ) and hydroxylysine (Hyl, K * ). At present, more than 20 kinds of natural collagens have been isolated, which are called type I and type II collagen according to the order of discovery, and are distributed in animal skin, bones, ligaments, v...

Examples

Embodiment 1

[0033] This embodiment provides a long-acting recombinant type III collagen expressed by Saccharomyces cerevisiae, the amino acid sequence of the collagen includes basic repeating units and terminal amino acid sequences; the basic repeating units include human type III collagen peptides, human type III collagen The amino acid sequence of the peptide is GERGAPGFRGPAGPNGIPGEKGPAGERGAP; the terminal amino acid sequence includes the human type II collagen peptide, and the amino acid sequence of the human type II collagen peptide is GPPGPCCGGG.

[0034] The nucleotide sequence of the long-acting recombinant type III collagen includes a Not I restriction site, an Xba I restriction site, a start codon, a stop codon and a 6×His tag sequence. The nucleotide sequence of the Not I restriction site is GCGGCCGC; the nucleotide sequence of the Xba I restriction site is TCTAGA; the nucleotide sequence of the start codon is ATG; the nucleotide sequence of the stop codon is TAA .

[0035] The...

Embodiment 2

[0043] This example provides a method for detecting the activity of the long-acting recombinant type III collagen, which is used to verify the cell proliferation-promoting activity of the long-acting recombinant type III collagen described in Example 1. The cell line used in this detection method is BALB / C 3T3 cells.

[0044] The activity detection method includes reagent preparation, assay operation and assay method. Reagents include complete culture solution, maintenance culture solution, digestion solution, thiazolium blue solution, and PBS buffer. Determination operations include the preparation of recombinant human collagen test samples.

[0045] The assay method comprises the following steps:

[0046] Step 1, the cultivation of the cell line, the cell line is BALB / C 3T3 cell line;

[0047] Step 2, preparing a cell suspension, inoculating the cell suspension on a 96-well plate;

[0048] Step 3, maintaining cell survival with maintenance medium;

[0049] Step 4, addin...

Embodiment 3

[0070] This example provides an application of Saccharomyces cerevisiae expressing long-acting recombinant type III collagen in cosmetics, which uses the long-acting recombinant type III collagen as described in Example 1. The long-acting recombinant type III collagen has good biocompatibility, good cell adhesion, can promote the formation of new cells, and has hemostatic function. And as described in Example 2, the long-acting recombinant type III collagen in Example 1 has a stronger function of promoting the growth of epithelial cells. It can be seen that the long-acting recombinant type III collagen described in Example 1 has broad application prospects in cosmetics.