CN114539389B - Recombinant collagen and application thereof - Google Patents
Recombinant collagen and application thereof Download PDFInfo
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- CN114539389B CN114539389B CN202210164104.0A CN202210164104A CN114539389B CN 114539389 B CN114539389 B CN 114539389B CN 202210164104 A CN202210164104 A CN 202210164104A CN 114539389 B CN114539389 B CN 114539389B
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- recombinant collagen
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Abstract
The invention discloses a recombinant collagen and application thereof, wherein the amino acid sequence of the recombinant collagen is (GAPGPRGAPGPAGPKGSA) m M is an integer more than or equal to 3, the amino acid sequence of the collagen is rich in arginine and lysine, and sufficient carboxyl and amino are provided for crosslinking, so that the collagen can be fully crosslinked under simple conditions, the crosslinking difficulty is greatly reduced, and the recombined collagen keeps the amino acid sequence of the collagen in tissue repair,The functions of moisture retention, water locking and the like are easier to be used in tissue materials and medical instruments.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to recombinant collagen and application thereof.
Background
Collagen is a biological high molecular protein, is a main component in animal connective tissues, is also a functional protein with the largest content and the widest distribution in mammals, and accounts for 25 to 30 percent of the total amount of protein. Has close relation with the formation and maturation of tissues, the transmission of intercellular information, joint lubrication, wound healing, calcification, blood coagulation, aging and the like, is one of the most key raw materials of the biotechnology industry, and is widely applied to the medical materials, cosmetics and food industry.
The collagen used industrially at present is mainly extracted from the skin or bone of an animal by an acid, alkali or enzymatic method, and the main source thereof is animal connective tissue, but the collagen extracted from animal tissue has risks such as animal-derived diseases and the like, and large-scale production causes enormous pressure on animal feeding on the supply side.
With the large-scale application of the genetic engineering technology, the bottleneck of large-scale preparation of collagen is successfully solved by using a modeling expression host to express the collagen in an exogenous protein expression mode in the genetic engineering recombinant expression. Compared with the traditional extraction of animal collagen, the preparation of collagen by genetic engineering recombination comprises the following steps: 1. the produced collagen has various types, can be common collagen from cattle, pigs, fishes and the like, can also be human collagen, and has better safety and immune efficiency in the fields of medical instruments and the like. 2. Animal-derived diseases can be effectively avoided, the recombinant collagen usually takes simple prokaryotic or eukaryotic cells as expression hosts, and the cellular pathogenic sources of the recombinant collagen cannot spread with human cells due to great difference of cellular structures. 3. The production period is short, the cost is saved, the scale-up can be quickly realized, and the method is suitable for industrial large-scale production. Compared with the method that the animal culture has the Merlot of 3-5 months, the method has the advantages that the microbial culture period is only 2-3 days, the culture is simple, the large-scale culture can be realized by providing simple C sources and N sources, and the industrial preparation is easy to carry out. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is the hot spot of the current collagen production research.
The amino acid composition of the recombinant human collagen is similar to or consistent with that of a human body, so that the recombinant human collagen is considered to be closest to the collagen naturally synthesized by the human body, has good biocompatibility and high safety, has the efficacy function of animal collagen, has a large number of literature reports, and has been successfully developed and applied to the fields of beauty, cosmetics and medical appliances. The currently reported recombinant collagen mainly focuses on the recombinant expression of a human full-length single-chain and the recombinant expression of a human collagen functional region repeat fragment, and compared with the recombinant expression of the full-length single-chain expression functional region repeat fragment, the recombinant collagen has the following characteristics: 1) The target protein has small molecular weight, can be expressed in various expression systems in high yield, and is easy to realize large-scale production; 2) The structure is stable, the acid and alkali resistance and the temperature capability are strong, and the separation and purification are easy; 3) The recombinant collagen has the effects of promoting skin surface cell repair, keeping full long chains such as moisture and the like and the natural collagen, so the recombinant expression of the repeated segments of the functional regions is a common preparation form of the recombinant collagen at present.
The cross-linking of the recombinant collagen to form macromolecules with different molecular weights so as to form a product with a net-shaped and regular spatial structure is an important application field of the product, and the product is applied to tissue engineering and medical instruments, however, the cross-linking of most of the recombinant collagen is realized by adopting a plurality of means due to the characteristic of amino acid composition in a functional region, which has the defects of few cross-linking sites and difficult realization of high cross-linking degree, and the removal of a cross-linking agent after the realization of a plurality of cross-linking means becomes a restriction bottleneck of subsequent application, so that a novel recombinant collagen which is simple and easy to realize and a preparation method are urgently needed, the cross-linking site occupation ratio is improved from the aspect of protein amino acid composition, the difficulty of subsequent cross-linking is reduced, and various physiological activities of the recombinant collagen can be reserved.
Disclosure of Invention
Aiming at the defects existing in the cross-linking process of the recombinant collagen in the prior art, the invention provides the collagen, the ratio of arginine to lysine in an amino acid sequence is effectively improved by optimizing the amino acid composition on the basis of the classic G-X-Y structure of the collagen, and compared with the existing collagen, the recombinant collagen is easier to realize cross-linking, has good structural stability after cross-linking, and can be better used in collagen-based medical instruments.
The specific technical scheme of the invention is as follows:
1. a recombinant collagen, the amino acid sequence of the recombinant collagen is (GAPGPRGAPGPAGPKGSA) m And m is an integer not less than 3.
2. The recombinant collagen according to claim 1, wherein m is an integer < 100, preferably 3 to 50, and more preferably m =10, 14 or 28.
3. A nucleic acid molecule, wherein said nucleic acid molecule encodes the recombinant collagen of item 1 or 2.
4. A vector, wherein said vector comprises the nucleic acid molecule of item 3.
5. The vector according to item 4, wherein the vector is an exogenous plasmid or an autosome.
6. A host cell comprising the vector of item 4 or 5.
7. The host cell according to claim 6, wherein the host cell is a prokaryotic cell or a eukaryotic cell, preferably the prokaryotic cell is Escherichia coli, bacillus subtilis or Bacillus licheniformis, preferably Escherichia coli;
preferably, the eukaryotic cell is pichia pastoris, saccharomyces cerevisiae, animal cells or plant cells, and is preferably pichia pastoris.
8. A method of producing the recombinant collagen of any one of claims 1-2, comprising the steps of:
the host cell according to item 6 or 7 is expressed, and then isolated and purified.
9. The method of item 8, wherein said expression is constitutive expression and/or inducible expression.
10. The method according to item 8 or 9, wherein the separation and purification is performed by one or more methods selected from the group consisting of salting-out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably by a combination of chromatography and membrane separation, and more preferably by a combination of ion exchange chromatography and membrane separation.
11. Use of the recombinant collagen of any one of items 1-2 in a skin care product, a health care product, a tissue material or a medical device, preferably in a tissue material or a medical device.
12. The use of item 11, wherein the recombinant collagen is in the form of macromolecules formed by cross-linking.
13. A cross-linked recombinant collagen, wherein the cross-linked recombinant collagen is obtained by cross-linking the recombinant collagen of item 1 or 2 or the recombinant collagen encoded by the nucleic acid molecule of item 3 or the recombinant collagen expressed by the vector of item 4 or 5 or the cross-linked protein produced by the host cell of item 6 or 7.
14. The recombinant collagen of claim 13, wherein the catalyst that catalyzes cross-linking is transglutaminase or carbodiimide.
15. The crosslinked recombinant collagen according to claim 13 or 14, wherein the degree of crosslinking of the crosslinked recombinant collagen is 90% or more.
16. A collagen sponge obtained by freeze-drying the crosslinked recombinant collagen of any one of items 13 to 15.
ADVANTAGEOUS EFFECTS OF INVENTION
The amino acid sequence of the collagen provided by the invention is rich in arginine and lysine, so that sufficient carboxyl and amino groups are provided for crosslinking, the collagen can be fully crosslinked under simple conditions, the crosslinking difficulty is greatly reduced, and meanwhile, the recombined collagen retains the functions of the collagen in the aspects of tissue repair, moisture retention, water locking and the like, and is more easily applied to tissue materials and medical instruments.
Drawings
FIG. 1 is a schematic diagram of electrophoresis after expression using E.coli in example 1-1, wherein lane 1 is a schematic diagram of crude protein expression solution after wall breaking, lane 2 is a schematic diagram of host blank, M is low molecular marker, and HLC is recombinant collagen.
FIG. 2 is a schematic diagram of electrophoresis after expression using yeast in example 1-2, wherein lane 1 is a schematic diagram of fermentation supernatant, lane 2 is a schematic diagram of yeast host blank, M is low molecular marker, HLC is recombinant collagen.
FIG. 3 is a schematic diagram of electrophoresis of 10 repeats of a purified recombinant protein, wherein M is a low molecular weight marker.
FIG. 4 is a schematic electrophoresis diagram of 14 repeats of the purified recombinant protein, wherein M is low molecular weight marker.
FIG. 5 is a schematic electrophoresis diagram of the purified 28 repeats of recombinant protein, wherein M is low molecular weight marker.
Detailed Description
The embodiments described below explain the present invention in detail. While specific embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and following claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the invention is to be determined by the claims appended hereto.
The invention provides a recombinant collagen, the amino acid sequence of which is (GAPGPRGAPGPAGPKGSA) m And m is an integer not less than 3.
The amino acid sequence of the recombinant collagen is formed by optimizing amino acid on the basis of the recombinant collagen disclosed in claim 1 of Chinese patent publication CN1371919A and on the basis of keeping the classic G-X-Y structure of the collagen, so that the ratio of arginine to lysine in the amino acid sequence is effectively increased.
Wherein GAPGPRGAPGPAGPKGSA is SEQ ID NO:1.
m can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and the like.
In one embodiment, m is an integer < 100, preferably 3 to 50, more preferably m =10, 14 or 28.
Wherein, when m =10, the amino acid sequence is shown in SEQ ID NO. 2, and the amino acid sequence is:
GAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPG PAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGP RGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSA GAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSA
when m =14, the amino acid sequence is shown as SEQ ID NO. 3, and the amino acid sequence is as follows:
GAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPG PAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGP RGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSA GAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAG PKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGA PGPAGPKGSA
when m =28, the amino acid sequence is shown as SEQ ID NO. 4, and the amino acid sequence is as follows:
GAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPG PAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGP RGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSA GAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAG PKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGA PGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAP GPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKG SAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGP AGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPR GAPGPAGPKGSAGAPGPRGAPGPAGPKGSAGAPGPRGAPGPAGPKGSAG APGPRGAPGPAGPKGSA
the invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the recombinant collagen.
In one embodiment, when m =10, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No. 5, which nucleotide sequence is:
ggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccggg cccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgcc gggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggccc gaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcggg cgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcg cggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggccc ggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaagg cagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcg
when m =14, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO 6, and the nucleotide sequence is as follows:
ggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcg ccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggc ccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgc ggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcg ccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggc ccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcg ggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccg aaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggc agcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcg ggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcg ccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggc ccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgc ggcgcgccgggcccggcgggcccgaaaggcagcgcg
when m =28, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:7, and the nucleotide sequence is as follows:
ggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccggg cccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgcc gggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggccc gaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcggg cgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcg cggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggccc ggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaagg cagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgcc gggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgc gccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcggg cccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgc gggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggccc gcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccggg cccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaa aggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgc gccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcgg cgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggc gggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcag cgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccggg cccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgcc gggcccggcgggcccgaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggccc gaaaggcagcgcgggcgcgccgggcccgcgcggcgcgccgggcccggcgggcccgaaaggcagcgcg
in some cases, the nucleic acid molecule encoding the recombinant collagen can be obtained from a variety of sources, such as by Polymerase Chain Reaction (PCR) amplification of the encoding nucleic acid within or isolated from one or more given cells.
In one embodiment, the nucleotide sequence encoding the recombinant collagen protein is codon optimized. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between the transfected gene and the host cell may affect protein expression and immunogenicity of the nucleic acid construct. Typically, for codon optimization, codons are selected to select those codons that are balanced with host frequency of use. Typically, the redundancy of amino acid codons is such that different codons encode one amino acid.
The present invention provides a vector comprising the nucleic acid molecule as described above.
In one embodiment, the vector is an exogenous plasmid or an autosome.
For example, one or more nucleic acids encoding the recombinant collagen proteins described above are cloned into a suitable expression vector or vectors, which may be any suitable recombinant expression vector, and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific for the type of host into which the vector is to be introduced (e.g., bacterial, fungal, plant or animal), as appropriate and taking into account whether the vector is DNA-based or RNA-based.
The present invention provides a host cell comprising the vector described above.
To produce recombinant collagen, the nucleic acid encoding the recombinant collagen can be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding recombinant collagen).
The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell.
Methods for introducing a vector into a host cell are well known, and for example, a vector is introduced into a host cell by heat shock.
In one embodiment, the host cell is a prokaryotic or eukaryotic cell, preferably the prokaryotic cell is escherichia coli, bacillus subtilis or bacillus licheniformis, preferably escherichia coli;
preferably, the eukaryotic cell is pichia pastoris, saccharomyces cerevisiae, animal cells or plant cells, and is preferably pichia pastoris.
The invention provides a method for preparing the recombinant collagen, which comprises the following steps:
the host cell is expressed and then separated and purified to obtain the recombinant DNA.
The expression of the host cell means that the host cell is cultured in a medium and culture conditions known to those skilled in the art, and for example, the host cell is cultured in a medium such as LB, YPD, YNB or the like.
The expression mode is not limited, and the expression mode can be confirmed according to requirements, such as constitutive expression or inducible expression or combination of the constitutive expression and the inducible expression, the inducer can be IPTG, beta-galactoside, methanol, ethanol and the like.
The method of separation and purification is not limited in any way, and may be determined as needed, and for example, one or a combination of two methods such as salting-out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation may be used, preferably a combination of chromatography and membrane separation, and more preferably a combination of ion exchange chromatography and membrane separation.
In one embodiment, the recombinant collagen is efficiently expressed and produced in an expression host by means of molecular cloning, and preferably, the molecular cloning means is a method for efficiently producing recombinant collagen by utilizing the efficient protein synthesis characteristics of a host cell itself in such a manner that a gene sequence of the recombinant collagen is carried on a plasmid and integrated into a host chromosome.
The amino acid sequence of the recombinant collagen provided by the invention is rich in arginine and lysine, so that sufficient carboxyl and amino are provided for crosslinking, the collagen can be fully crosslinked under simple conditions, and the crosslinking difficulty is greatly reduced.
The invention provides application of the collagen in skin care products, health care products, tissue materials or medical devices, preferably application in the tissue materials or the medical devices.
In one embodiment, the recombinant collagen is in the form of a macromolecule formed by cross-linking.
According to the invention, by using the recombinant collagen, the amino acid sequence is rich in arginine and lysine, which provides sufficient carboxyl and amino groups for crosslinking, so that the collagen is easier to crosslink, and meanwhile, the recombinant collagen retains the functions of the collagen in the aspects of tissue repair, moisture retention, water locking and the like, and is easier to be used in tissue materials and medical instruments.
The invention provides a cross-linked recombinant collagen, which is obtained by cross-linking the recombinant collagen or the recombinant collagen coded by the nucleic acid molecule or the recombinant collagen expressed by the vector or the cross-linked protein produced by the host cell.
The crosslinking refers to the process of connecting small molecular proteins or linear proteins into net-shaped or high molecular proteins by covalent bonds.
In one embodiment, the catalyst that catalyzes the crosslinking is transglutaminase, EDC (carbodiimide), or the like.
In one embodiment, the cross-linking degree of the cross-linked recombinant collagen is 90% or more.
The invention provides a collagen sponge, which is obtained by freeze-drying the crosslinked recombinant collagen.
As for the freeze drying method, it is a method known to those skilled in the art, for example, the cross-linked recombinant collagen is freeze-dried in a freeze dryer, preferably at-40 deg.C for 12h.
Examples
The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments are not indicated by manufacturers, and are conventional reagent products which can be obtained commercially, wherein the enzymes used are all purchased from TaKaRa company, the plasmid DNA extraction kit and the DNA gel recovery kit are all purchased from Beijing Solibao company, and the gene recombination kit (recombination Kits) is purchased from Tiangen organisms, and the specific operation completely follows the instruction of the kit.
Examples 1-1m =10 preparation of recombinant collagen
A. Preparation of recombinant collagen using E.coli expression System (m = 10)
1. Construction of expression Strain
Construction of pKK 223-3-NHLC: will be as shown in SEQ ID No:2 according to the codon preference of Escherichia coli, synthesizing by a whole-gene synthesis mode to obtain a nucleotide sequence shown as SEQ ID NO. 5, then directly connecting the nucleotide sequence to pKK223-3 plasmid to obtain pKK223-3-NHLC, transferring the pKK223-3-NHLC into DE3 competent cells (DE 3 competent cells purchased by Shanghai life worker), coating a resistant plate, and selecting a single colony on the plate as an expression strain.
2. Inducible expression of target proteins
A single colony of the expression strain was picked and inoculated into a tube containing 2.5ml of LB medium (supplemented with ampicillin), and cultured overnight at 37 ℃. Transferring the seed solution cultured overnight into 100ml LB liquid culture medium with 1% inoculum size, culturing at 37 ℃,200rpm until the OD value is about 2-3, adding IPTG with the final concentration of 1.5mM, cooling to 28 ℃ for induction culture, inducing for 14h, centrifugally collecting thalli, preparing 10% (thalli wet weight/PB volume) bacterial suspension with PB buffer solution with the pH of 6.0, homogenizing for 3min under high pressure under the condition of 1000bar, centrifugally collecting supernatant, namely crude protein expression solution, and identifying by an electrophoresis method: and (3) taking competent cells DE3 without introducing plasmids as an induction control, breaking the walls of the control group and the constructed expression strain group at the same time after the induction is finished, and detecting the SDSPAGE protein by electrophoresis, wherein the result is shown in figure 1.
It can be seen from fig. 1 that the bands significantly more than the control group are the target bands.
B. Production of recombinant collagen in a yeast expression System
Construction of pPIC9k plasmid
Extracting pKK223-3-NHLC plasmid obtained in A, performing double enzyme digestion on the pPIC 223-3-NHLC plasmid by using SnaBI and NotI enzymes to recover a fragment of about 756, connecting the fragment with pPIC9k which is also subjected to enzyme digestion by the SnaBI and the NotI enzymes under the action of T4 ligase, connecting overnight at 16 ℃, transferring the obtained product into Top10 competent cells, coating a bleomycin resistant plate, screening transformants, performing double enzyme digestion by using the SnaBI and the NotI enzymes, obtaining transformants which can generate 9700kb and 756bp bands after enzyme digestion, namely positive transformants, and extracting the positive plasmids to obtain the pPIC9k-NHLC transformants.
2. Preparation of Yeast expression strains
And linearizing pPIC9k-NHLC by Sac I, transforming pichia pastoris GS115 competence, and screening high-copy transformants by taking G418 resistance as a screening marker to obtain the yeast expression strain.
3. Inducible expression of target proteins
(1) Picking single colony of co-expression strain, adding into 5ml YPD liquid culture medium (1% yeast extract, 2% peptone and 2% glucose), culturing at 30 deg.C and 200rpm overnight for activation;
(2) Inoculating 1% of the strain into 100ml of BMGY liquid medium, and culturing at 30 ℃ and 200rpm until the OD600= 6.0-9.0;
(3) Centrifuging at 25 deg.C for 6min under the action of 1500g centrifugal force, collecting thallus, suspending in 200ml BMMY liquid culture medium to make its initial concentration OD600=1.0, and culturing at 30 deg.C and 200 rpm;
(4) Adding methanol every 24h to make induction expression, its final concentration is 0.5-1.0%;
(5) Inducing for 72h, taking the culture solution, centrifuging for 2min under the condition of 12000rpm, and taking the supernatant.
4. Recombinant collagen purification
Adjusting pH of 30ml of crude protein expression solution to 6.0, performing crude purification by using 1ml of capto S prepacked column of GE company as a purification medium, separating the crude purified sample by using a molecular sieve, and performing ultrafiltration concentration to obtain the recombinant collagen with the purity of 90%, wherein the recombinant collagen is identified by an electrophoresis method: taking yeast expression strains without plasmids as induction control, breaking cell wall of the control group and the constructed expression strain group after induction, detecting SDSPAGE protein electrophoresis, and obtaining the result shown in figure 2 and the purified electrophoresis image shown in figure 3.
It can be seen from fig. 2 that the bands significantly more than the control group are the target bands.
Example 1-2m =14 preparation of recombinant collagen
The recombinant collagen was prepared in the same manner as in example 1-1, and the electrophoretogram after purification was shown in FIG. 4, in which the nucleotide sequence was SEQ ID No. 6 and the amino acid sequence was SEQ ID No. 3.
Example 1-3m =28 preparation of recombinant collagen
The recombinant collagen was prepared in the same manner as in example 1-1, and the electrophoretogram after purification was shown in FIG. 5, in which the nucleotide sequence was SEQ ID 7 and the amino acid sequence was SEQ ID NO. 4.
EXAMPLE 2-1 preparation of collagen sponge
Freeze-drying the purified collagen prepared in example 1-1 into dry powder, accurately weighing refined TG enzyme (Shanghai source leaf, 25 g/bottle, 0.005%), dispersing in a certain volume of purified water, fully dissolving, weighing collagen freeze-dried powder (2%), stirring in a stirrer at room temperature (25 ℃) until fully dissolving, standing for defoaming, filling and dispersing in a mold;
filling the filled collagenThe solution is placed in a refrigerator at the temperature of 2-8 ℃ and is kept stand for 48 hours, the crosslinking degree of the recombinant collagen obtained after crosslinking is measured by an indetrione method, and the specific operation method comprises the following steps: crosslinking degree measurement (indetrione method): determination of the concentration C of free amino acids before and after crosslinking by the Indantrione method Front side And C Rear end The crosslinking effect is reacted through the change of the amino acid concentration before and after crosslinking:
a (degree of crosslinking) = (C) Front- C Rear end )/C Front X100%
The crosslinking degree is 99%; then putting the crosslinked collagen sponge into a freeze dryer (LYO-0.5) for freeze-drying, and freeze-drying at-40 deg.C overnight to obtain the collagen sponge.
Example 2-2 preparation of collagen sponge
The recombinant collagen obtained in example 1-2 was crosslinked in the same manner as in example 2-1 to obtain a recombinant collagen having a degree of crosslinking of 98%, and a collagen sponge was prepared in the same manner as in example 2-1.
Examples 2-3 preparation of collagen sponge
The recombinant collagen obtained in example 1-2 was crosslinked in the same manner as in example 2-1 to obtain a recombinant collagen having a crosslinking degree of 96%, and a collagen sponge was prepared in the same manner as in example 2-1.
Example 3 comparison of the degree of crosslinking before and after optimization
The recombinant collagens obtained in examples 1-1 to 1-3 and the unoptimized collagen were cross-linked, and the cross-linking degree was determined by the same method as in example 2-1, wherein the amino acid sequence of the unoptimized collagen was shown as SEQ ID No. 8 when m =14, as SEQ ID No. 9 when m =28, and as SEQ ID No. 10 when m =10, wherein the amino acid sequence shown as SEQ ID No. 8 is as follows:
GAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGP AGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPG APGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAP GPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSA GAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPP GSA
the amino acid sequence shown in SEQ ID NO. 9 is as follows:
GAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGP AGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPG APGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAP GPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSA GAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPP GSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGP AGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPG APGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAP GPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSA GAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPP GSA
the amino acids shown in SEQ ID NO. 10 are as follows:
GAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGP AGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPG APGPAGPPGSAGAPGPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSAGAP GPPGAPGPAGPPGSAGAPGPPGAPGPAGPPGSA
the measurement results are shown in table 1.
TABLE 1 comparison of the degree of crosslinking
| Example 3 | Protein of non-optimized amino acid | |
| m＝14 | 98％ | 80％ |
| m＝28 | 96％ | 75％ |
| m＝10 | 99％ | 85％ |
As can be seen from the above table, the recombinant collagen provided by the invention is rich in arginine and lysine, is easy to crosslink, simultaneously retains the functions of the collagen in the aspects of tissue repair, moisture retention, water locking and the like, and is easier to be used in tissue materials and medical instruments.
In conclusion, the recombinant collagen provided by the invention is rich in arginine and lysine, is easy to crosslink, can realize sufficient crosslinking under simple conditions, retains the functions of the collagen in the aspects of tissue repair, moisture retention, water locking and the like, and is easier to apply to tissue materials and medical instruments.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Sequence listing
<110> Shaanxi Dazi Biotechnology Co., ltd
<120> recombinant collagen and use thereof
<130> TPE01521
<160> 10
<170> PatentIn version 3.5
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Gly Ala Pro Gly Pro Arg Gly Ala Pro Gly Pro Ala Gly Pro Lys Gly
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ggcgcgccgg gcccgcgcgg cgcgccgggc ccggcgggcc cgaaaggcag cgcgggcgcg 60
ccgggcccgc gcggcgcgcc gggcccggcg ggcccgaaag gcagcgcggg cgcgccgggc 120
ccgcgcggcg cgccgggccc ggcgggcccg aaaggcagcg cgggcgcgcc gggcccgcgc 180
ggcgcgccgg gcccggcggg cccgaaaggc agcgcgggcg cgccgggccc gcgcggcgcg 240
ccgggcccgg cgggcccgaa aggcagcgcg ggcgcgccgg gcccgcgcgg cgcgccgggc 300
ccggcgggcc cgaaaggcag cgcgggcgcg ccgggcccgc gcggcgcgcc gggcccggcg 360
ggcccgaaag gcagcgcggg cgcgccgggc ccgcgcggcg cgccgggccc ggcgggcccg 420
aaaggcagcg cgggcgcgcc gggcccgcgc ggcgcgccgg gcccggcggg cccgaaaggc 480
agcgcgggcg cgccgggccc gcgcggcgcg ccgggcccgg cgggcccgaa aggcagcgcg 540
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ggcgcgccgg gcccgcgcgg cgcgccgggc ccggcgggcc cgaaaggcag cgcgggcgcg 60
ccgggcccgc gcggcgcgcc gggcccggcg ggcccgaaag gcagcgcggg cgcgccgggc 120
ccgcgcggcg cgccgggccc ggcgggcccg aaaggcagcg cgggcgcgcc gggcccgcgc 180
ggcgcgccgg gcccggcggg cccgaaaggc agcgcgggcg cgccgggccc gcgcggcgcg 240
ccgggcccgg cgggcccgaa aggcagcgcg ggcgcgccgg gcccgcgcgg cgcgccgggc 300
ccggcgggcc cgaaaggcag cgcgggcgcg ccgggcccgc gcggcgcgcc gggcccggcg 360
ggcccgaaag gcagcgcggg cgcgccgggc ccgcgcggcg cgccgggccc ggcgggcccg 420
aaaggcagcg cgggcgcgcc gggcccgcgc ggcgcgccgg gcccggcggg cccgaaaggc 480
agcgcgggcg cgccgggccc gcgcggcgcg ccgggcccgg cgggcccgaa aggcagcgcg 540
ggcgcgccgg gcccgcgcgg cgcgccgggc ccggcgggcc cgaaaggcag cgcgggcgcg 600
ccgggcccgc gcggcgcgcc gggcccggcg ggcccgaaag gcagcgcggg cgcgccgggc 660
ccgcgcggcg cgccgggccc ggcgggcccg aaaggcagcg cgggcgcgcc gggcccgcgc 720
ggcgcgccgg gcccggcggg cccgaaaggc agcgcg 756
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ggcgcgccgg gcccgcgcgg cgcgccgggc ccggcgggcc cgaaaggcag cgcgggcgcg 60
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ccgcgcggcg cgccgggccc ggcgggcccg aaaggcagcg cgggcgcgcc gggcccgcgc 180
ggcgcgccgg gcccggcggg cccgaaaggc agcgcgggcg cgccgggccc gcgcggcgcg 240
ccgggcccgg cgggcccgaa aggcagcgcg ggcgcgccgg gcccgcgcgg cgcgccgggc 300
ccggcgggcc cgaaaggcag cgcgggcgcg ccgggcccgc gcggcgcgcc gggcccggcg 360
ggcccgaaag gcagcgcggg cgcgccgggc ccgcgcggcg cgccgggccc ggcgggcccg 420
aaaggcagcg cgggcgcgcc gggcccgcgc ggcgcgccgg gcccggcggg cccgaaaggc 480
agcgcgggcg cgccgggccc gcgcggcgcg ccgggcccgg cgggcccgaa aggcagcgcg 540
ggcgcgccgg gcccgcgcgg cgcgccgggc ccggcgggcc cgaaaggcag cgcgggcgcg 600
ccgggcccgc gcggcgcgcc gggcccggcg ggcccgaaag gcagcgcggg cgcgccgggc 660
ccgcgcggcg cgccgggccc ggcgggcccg aaaggcagcg cgggcgcgcc gggcccgcgc 720
ggcgcgccgg gcccggcggg cccgaaaggc agcgcgggcg cgccgggccc gcgcggcgcg 780
ccgggcccgg cgggcccgaa aggcagcgcg ggcgcgccgg gcccgcgcgg cgcgccgggc 840
ccggcgggcc cgaaaggcag cgcgggcgcg ccgggcccgc gcggcgcgcc gggcccggcg 900
ggcccgaaag gcagcgcggg cgcgccgggc ccgcgcggcg cgccgggccc ggcgggcccg 960
aaaggcagcg cgggcgcgcc gggcccgcgc ggcgcgccgg gcccggcggg cccgaaaggc 1020
agcgcgggcg cgccgggccc gcgcggcgcg ccgggcccgg cgggcccgaa aggcagcgcg 1080
ggcgcgccgg gcccgcgcgg cgcgccgggc ccggcgggcc cgaaaggcag cgcgggcgcg 1140
ccgggcccgc gcggcgcgcc gggcccggcg ggcccgaaag gcagcgcggg cgcgccgggc 1200
ccgcgcggcg cgccgggccc ggcgggcccg aaaggcagcg cgggcgcgcc gggcccgcgc 1260
ggcgcgccgg gcccggcggg cccgaaaggc agcgcgggcg cgccgggccc gcgcggcgcg 1320
ccgggcccgg cgggcccgaa aggcagcgcg ggcgcgccgg gcccgcgcgg cgcgccgggc 1380
ccggcgggcc cgaaaggcag cgcgggcgcg ccgggcccgc gcggcgcgcc gggcccggcg 1440
ggcccgaaag gcagcgcggg cgcgccgggc ccgcgcggcg cgccgggccc ggcgggcccg 1500
aaaggcagcg cg 1512
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Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
1 5 10 15
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
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Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
35 40 45
Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
50 55 60
Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala
65 70 75 80
Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro
85 90 95
Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly
100 105 110
Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala
115 120 125
Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala
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Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
145 150 155 160
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
165 170 175
Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
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Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
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Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala
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Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala
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<223> description of artificial sequences: artificially synthesized sequences
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Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
1 5 10 15
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
20 25 30
Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
35 40 45
Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
50 55 60
Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala
65 70 75 80
Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro
85 90 95
Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly
100 105 110
Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala
115 120 125
Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala
130 135 140
Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
145 150 155 160
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
165 170 175
Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
180 185 190
Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
195 200 205
Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala
210 215 220
Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro
225 230 235 240
Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly
245 250 255
Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala
260 265 270
Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala
275 280 285
Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
290 295 300
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
305 310 315 320
Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
325 330 335
Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
340 345 350
Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala
355 360 365
Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro
370 375 380
Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly
385 390 395 400
Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala
405 410 415
Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala
420 425 430
Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
435 440 445
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
450 455 460
Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
465 470 475 480
Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
485 490 495
Pro Ala Gly Pro Pro Gly Ser Ala
500
<210> 10
<211> 180
<212> PRT
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<220>
<223> Artificial sequence description: artificially synthesized sequences
<400> 10
Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
1 5 10 15
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
20 25 30
Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala
35 40 45
Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly
50 55 60
Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala
65 70 75 80
Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Pro Pro
85 90 95
Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly
100 105 110
Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala Gly Ala
115 120 125
Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly Ser Ala
130 135 140
Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro Pro Gly
145 150 155 160
Ser Ala Gly Ala Pro Gly Pro Pro Gly Ala Pro Gly Pro Ala Gly Pro
165 170 175
Pro Gly Ser Ala
180
Claims (23)
1. A recombinant collagen, the amino acid sequence of the recombinant collagen is (GAPGPRGAPGPAGPKGSA) m Wherein m =10, 14 or 28.
2. A nucleic acid molecule, wherein said nucleic acid molecule encodes the recombinant collagen of claim 1.
3. A vector, wherein said vector comprises the nucleic acid molecule of claim 2.
4. The vector of claim 3, wherein the vector is an exogenous plasmid.
5. A host cell comprising the vector of claim 3 or 4.
6. The host cell of claim 5, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
7. The host cell of claim 6, wherein the prokaryotic cell is E.coli, B.subtilis, or B.licheniformis.
8. The host cell of claim 6, wherein the prokaryotic cell is E.
9. The host cell of claim 6, wherein the eukaryotic cell is a Pichia, saccharomyces cerevisiae, or an animal cell.
10. The host cell of claim 6, wherein the eukaryotic cell is Pichia pastoris.
11. A method of preparing the recombinant collagen of claim 1, comprising the steps of:
the host cell of any one of claims 5-10, which is expressed and then isolated and purified.
12. The method of claim 11, wherein said expression is constitutive expression and/or inducible expression.
13. The method according to claim 11 or 12, wherein the separation and purification is performed by one or more methods selected from salting out, chromatography, acid-base precipitation, and membrane separation.
14. The method according to claim 11 or 12, wherein the method of separation and purification is a combination of chromatography and membrane separation.
15. The method according to claim 11 or 12, wherein the separation and purification method is a combination of ion exchange chromatography and membrane separation.
16. Use of the recombinant collagen of claim 1 in the preparation of a skin care product.
17. Use of the recombinant collagen of claim 1 for the preparation of a tissue material.
18. Use of the recombinant collagen of claim 1 for the manufacture of a medical device.
19. The use according to any one of claims 16-18, wherein the recombinant collagen is in the form of a macromolecule formed by cross-linking.
20. A cross-linked recombinant collagen, wherein the cross-linked recombinant collagen is obtained by cross-linking the recombinant collagen of claim 1, or the recombinant collagen encoded by the nucleic acid molecule of claim 2, or the recombinant collagen expressed by the vector of claim 3 or 4, or the recombinant collagen produced by the host cell of any one of claims 5 to 10.
21. The crosslinked recombinant collagen according to claim 20, wherein the catalyst for catalyzing the crosslinking is transglutaminase or carbodiimide.
22. The crosslinked recombinant collagen according to claim 20 or 21, wherein the degree of crosslinking of the crosslinked recombinant collagen is 90% or more.
23. A collagen sponge obtained by freeze-drying the cross-linked recombinant collagen of any one of claims 20-22.
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| CA2560161A1 (en) * | 2005-09-30 | 2007-03-30 | Nazrul Islam (Deceased) | Production of recombinant human collagen |
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| CA2560161A1 (en) * | 2005-09-30 | 2007-03-30 | Nazrul Islam (Deceased) | Production of recombinant human collagen |
| WO2021224316A1 (en) * | 2020-05-06 | 2021-11-11 | Gelita Ag | Expression of collagen peptide components in procaryotic systems |
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