CN111393522A

CN111393522A - Collagen and application thereof

Info

Publication number: CN111393522A
Application number: CN202010228673.8A
Authority: CN
Inventors: 秦亦如; 李胜; 杜春宇; 李颖
Original assignee: Guangmeiyuan R & D Center Key Laboratory Of Insect Developmental Biology And Applied Technology Huashi Meizhou City; South China Normal University
Current assignee: Guangmeiyuan R & D Center Key Laboratory Of Insect Developmental Biology And Applied Technology Huashi Meizhou City; South China Normal University
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2020-07-10
Anticipated expiration: 2040-03-27
Also published as: CN111393522B

Abstract

The invention relates to the field of biotechnology and medicine, in particular to collagen and application thereof. Specifically, the invention relates to an isolated collagen selected from the group consisting of: (a) 1, a collagen having an amino acid sequence of SEQ ID NO; or (b) collagen which is formed by substituting, deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO. 1 and has the function of promoting wound healing and is derived from the collagen (a). The collagen can rapidly and efficiently promote the healing of wounds and has a huge application prospect.

Description

Collagen and application thereof

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to an amino acid sequence, collagen coded by the amino acid sequence, a preparation method of the collagen and application of the collagen in preparing a wound healing medicament.

Background

Earthworm, also known as "earthworm," has been used as a Chinese medicine in China for two thousand years. According to the records of Ben Cao gang mu, over 40 prescriptions are combined with earthworm, and can be used for treating scrofula and ulceration, mouth toxicity and hot sores, mouth and tongue aphthae, etc. Modern earthworm separation technology discovers that earthworms contain various active ingredients such as highly unsaturated fatty acid, nucleotide, enzymes, proteins, earthworm thermokaline, fibrinolysin, antibacterial peptide, various antioxidant enzymes and the like, so that the earthworm separation technology has the effects of clearing heat, arresting convulsion, relieving cough and asthma, promoting blood circulation, removing obstruction in channels, reducing blood pressure, resisting ulcer, resisting hepatic fibrosis, promoting urination, stopping bleeding, easing pain, promoting wound healing, resisting oxidation, resisting thrombus, regulating immunity, resisting arrhythmia, resisting cancer and the like. In modern folk prescription, the earthworm and white sugar lixivium is also commonly used for smearing the wound surface, thereby improving the microcirculation of the affected part and treating scald, ulcer and the like.

In addition, earthworms have strong ability of regeneration and repair, and can rapidly heal wounds and regenerate bodies after being damaged. The external stimulation can induce the earthworm body to generate stress reaction, and active substances are generated to promote the repair of the damaged tissues and the regeneration of granulation. Research has shown that the extract of the earthworm with broken body has very good effect of promoting the healing of wound, but the effective component for promoting the healing of wound in the earthworm with broken body has not been reported so far.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. The invention provides collagen separated from earthworms, which has the function of promoting wound healing.

The first purpose of the invention is to provide collagen which can promote wound healing in earthworms.

It is a second object of the present invention to provide an amino acid sequence encoding said collagen.

It is a third object of the present invention to provide a method for preparing the collagen.

The fourth purpose of the invention is to provide the application of the collagen in preparing a wound healing medicament.

The technical scheme of the invention is as follows:

one aspect of the present invention provides an isolated collagen selected from the group consisting of:

(a) 1, a collagen having an amino acid sequence of SEQ ID NO; or

(b) 1 through one or more amino acid residue substitution, deletion and/or addition, and has the function of promoting wound healing.

According to some embodiments of the invention, the collagen has the amino acid sequence shown in SEQ ID NO 1.

The specific method of substitution, deletion or addition may be any method known in the art.

Through a large amount of scientific researches, the protein capable of efficiently promoting wound healing is separated from the corpus fibrosum earthworms for the first time. Specifically, the inventor finds that the broken earthworm extract (preferably 1-5 days of broken earthworms, and more preferably 2 days of broken earthworms) in Eisenia andrei has better wound healing effect than the unbroken earthworm extract, and performs mass spectrometry on the two extracts and combined analysis with up-regulated genes in a transcriptome, which indicates that the broken earthworm extract only contains the protein and is up-regulated on the transcriptome level; through Blast comparison analysis with an NIH database, the protein is confirmed to belong to earthworm collagen. The amino acid sequence of the protein is obtained by utilizing transcriptomics, a corresponding primer is synthesized on the basis of the amino acid sequence, a large amount of protein is obtained by in vitro expression of a prokaryotic expression system, and the protein is subjected to pharmacodynamic verification on NIH-3T3 to find that the protein has an obvious effect of promoting wound healing.

According to another aspect of the present invention there is provided an isolated collagen gene, the polynucleotide sequence of which comprises a nucleotide sequence encoding collagen as described above.

According to some embodiments of the invention, the polynucleotide encodes a collagen having the amino acid sequence shown in SEQ ID NO. 1.

According to some embodiments of the invention, the polynucleotide has the sequence shown in SEQ ID NO 2.

According to some embodiments of the invention, the polynucleotide may be a DNA nucleotide sequence of a gene mutant obtained by artificial site-directed mutagenesis of a gene encoding the collagen.

The polynucleotide of the present invention may be in the form of DNA or RNA, wherein the form of DNA includes cDNA, genomic DNA or synthetic DNA. The DNA may be single-stranded or double-stranded, and may be the coding strand or non-coding strand. The sequence of the coding region encoding the protein may be identical to that shown in SEQ ID NO. 2 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having SEQ ID NO. 1, but differ from the sequence of the coding region shown in SEQ ID NO. 2. The polynucleotide encoding the collagen of SEQ ID NO. 1 comprises: a coding sequence encoding only collagen; the coding sequence of collagen and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences of collagen.

The full-length polynucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the related sequences can be synthesized by artificial synthesis. At present, the DNA sequence encoding the collagen (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the collagen sequences of the present invention by chemical synthesis.

According to a further aspect of the present invention there is provided a recombinant vector comprising a polynucleotide sequence as described above.

According to some embodiments of the invention, the vector includes, but is not limited to, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, a retrovirus, or other vector; preferably, the vector is a bacterial plasmid.

The recombinant vectors of the present invention containing the polynucleotide sequences encoding the collagen proteins can be constructed using methods well known to those skilled in the art, including, but not limited to, in vitro recombinant DNA techniques, DNA synthesis techniques, in vitro recombinant techniques, and the like. The polynucleotide sequence may be operably linked to a suitable promoter in a vector to direct gene synthesis.

According to a further aspect of the present invention there is provided a host cell comprising a recombinant vector as described above.

According to some embodiments of the invention, the host cell may be a prokaryotic cell or a eukaryotic cell.

According to some embodiments of the invention, the host cell may be escherichia coli, yeast, insect-and mammalian-derived eukaryotic cell lines.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers, host cells, and the like.

Transformation of the recombinant vector into a host cell in the present invention can be carried out by conventional techniques well known to those skilled in the art. The obtained transformant can be cultured by a conventional method to express the collagen encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant protein in the above method may be expressed intracellularly, or on the cell membrane, or secreted extracellularly, if desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties, which are well known to those skilled in the art.

According to another aspect of the present invention, there is provided a method for preparing the collagen, comprising the steps of:

1) culturing a host cell as described above;

2) the collagen as described above was isolated from the culture.

According to some embodiments of the invention, the method for preparing collagen comprises the steps of:

1) amplifying a sequence shown as SEQ ID NO. 2, and connecting the sequence with a vector plasmid to obtain a recombinant vector;

2) transforming the recombinant vector obtained in the step 1) into a host cell;

3) culturing the host cell obtained in the step 2), collecting the induced cell, separating and purifying to obtain the collagen.

The collagen of the present invention may be a recombinant protein, a natural protein or a synthetic protein, preferably a recombinant protein; the collagen of the present invention may be a naturally purified product, or a chemically synthesized product, or obtained using a protein expression system using recombinant techniques, preferably a protein expression system.

According to some embodiments of the invention, in step 1), the primer sequences used for amplification are SEQ ID NO 3 and SEQ ID NO 4.

The primers used for amplification in the present invention can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.

According to a further aspect of the present invention there is provided the use of a collagen as described above in the manufacture of a medicament for wound healing.

The isolated collagen of the present invention has a variety of uses including, but not limited to: can be directly used as medicine for accelerating wound healing; directly used as a medicament for treating diseases caused by low or lost functions of the similar collagen; for screening for antibodies, polypeptides or other ligands that promote or antagonize the function of the collagen. Screening of polypeptide libraries with expressed recombinant collagen can be used to find therapeutically valuable polypeptide molecules that inhibit or stimulate the function of the collagen.

According to another aspect of the present invention, there is provided an antibody capable of specifically binding to the collagen.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptides encoded by the DNA of the collagen or fragments thereof. As used herein, "specificity" refers to the ability of an antibody to bind to the collagen gene product or fragment. Preferably, those antibodies that bind to the collagen gene product or fragment but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the collagen, as well as those antibodies that do not affect the collagen function. The invention also includes those antibodies which bind to the gene product of said collagen in modified or unmodified form. The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. The antibodies of the invention include antibodies that block the function of the collagen as well as antibodies that do not affect the function of the collagen. The various antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of the collagen gene product. The antibodies of the invention are useful for treating or preventing a disease associated with the collagen, and the administration of an appropriate amount of the antibodies can stimulate or block the production or activity of the collagen.

According to a further aspect of the invention there is provided a composition comprising a collagen as described above, a polynucleotide as described above, a recombinant vector as described above, a host cell as described above or an antibody as described above.

The inventor finds that the collagen separated from the extract of the tubificidae can safely and effectively promote the healing of the wound through research, and the separated collagen can be used as an active component alone or in combination with other active components. When the composition of the present invention is used, the specific dose can be appropriately adjusted depending on factors such as the administration route, individual differences, and the like.

According to some embodiments of the invention, the composition further comprises a pharmaceutical excipient.

The pharmaceutic adjuvant is a conventional pharmaceutic carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutic adjuvant; preferably, pharmaceutically acceptable disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants or preservatives are included.

As used herein, the term "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide, polypeptide or protein in the natural state in the living cell is not isolated or purified, the same polynucleotide, polypeptide or protein is isolated or purified if it is separated from other substances coexisting in the natural state.

As used herein, the term "collagen" refers to a protein or polypeptide having the amino acid sequence of SEQ ID NO. 1. The term also includes variants of the sequence of SEQ ID NO. 1 which have the same function as the collagen protein. These variants include (but are not limited to): substitution, deletion and/or addition of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids having similar or analogous properties will not generally alter the function of the polypeptide. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and/or active derivatives of the collagen.

As used herein, the term "polynucleotide encoding collagen" may include a polynucleotide encoding the collagen, and may also include additional coding and/or non-coding sequences.

The invention has the beneficial effects that:

the invention separates a new collagen from the complex extract of the earthworm with broken body for the first time, and the collagen can rapidly and efficiently promote the healing of the wound and has great application prospect.

Drawings

FIG. 1 is a sectional microscope image of the wound surface of earthworm on different days;

FIG. 2 is a graph showing the scratch repair effect of different earthworm extracts on mouse NIH-3T3 fibroblasts;

FIG. 3 is a graph showing the effects of different drugs on the repair of scratches on mouse NIH-3T3 fibroblasts at different times;

FIG. 4 is a graph showing the effect of different drugs on the repair of skin defects in mice at different times;

FIG. 5 HE staining of wound tissue in mice;

FIG. 6 is a transcriptomics up-and down-regulation gene difference volcano plot of 2-day-fragmented and non-fragmented earthworms;

FIG. 7 is a functional enrichment analysis graph of transcriptomics KEGG of earthworms with two days of fragmentation and earthworms without fragmentation;

FIG. 8 is a graph of mass spectra of earthworm extracts from two days after fragmentation, mass spectra of earthworm extracts from non-fragmental plants, and a Wein gene, a key enrichment gene in transcriptomics;

FIG. 9 is a mass spectrum of collagen;

FIG. 10 is a microscopic image of the scratch repair promotion effect of collagen and PBS on mouse NIH-3T3 fibroblasts at different times.

Detailed Description

The technical solutions of the present invention are further described below with reference to specific examples, but the present invention is not limited to these specific embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1 preparation of earthworm extract

After mud is spitted out in water of live earthworms of Eisenia andrei, the live earthworms are cleaned by deionized water, and the water on the surfaces of the earthworms is sucked by filter paper. 500g of earthworms with similar sizes are taken, and the earthworms are cut off at the position of 30-35 knots after the earthworm link by using a sterile scalpel blade under the ice bath condition. The broken earthworms are randomly and evenly divided into 5 groups and put back into the soil for feeding for 1 to 5 days. Taking out each earthworm sample, taking a picture under a microscope, cleaning, weighing, and adding ultrapure water according to the mass-volume ratio of 1: 1. The earthworms are crushed for 30 minutes by the aid of ultrasound, and then are put into a shaking table at the temperature of 4 ℃ at the speed of 200 rpm for homogenate extraction for 24 hours. Vacuum filtering, removing solid residue, and freeze drying the filtrate for 48 hr to obtain solid extract.

FIG. 1 is a sectional microscope image of the wound surface of earthworm section on different days. It can be seen that immediately after the incision (day 0), the wound surface was fresh and the body cavity was exposed as shown in a) in fig. 1. And within 1-2 days, muscle tissues on the earthworm sections are strengthened to contract, muscle cells are dissolved to form new cell masses, and meanwhile white blood cells in blood are gathered on the sections to form special embolism, so that the wound is closed, as shown in a b mode in the figure 1). At the fifth day, a clearly bud-like structure is formed in the section, and during this process, the cells of the tissues of the digestive tract, nervous system, blood vessels, etc. in the earthworm body grow rapidly into the regenerated bud through a large number of mitoses, see c) in fig. 1. In 1-2 days (wound healing period) after cutting, a large amount of active substances are secreted in the earthworms due to stimulation of external mechanical injury, and the substances can promote wound healing of the earthworms and have the potential capability of accelerating wound healing of mammals such as mice and the like.

Example 2 cell experiments of earthworm extract on wound repair in mice

During the period from mechanical injury stimulation of the operation of the amputation to granulation regeneration of wound healing, earthworms may secrete different active substances at different stages of the healing period, so that the extracts of earthworms at different days after the operation may have different drug effects. In order to find out the earthworm extract with the highest activity, the water extracts of earthworms 1 to 5 days after the amputation are respectively extracted in the embodiment of the invention, and the mechanical scratch cell repair model is firstly adopted to verify the repair effect on the wounds of mice.

Spreading a suitable number of mouse NIH-3T3 fibroblasts in each well of a 6-well plate; when the cell density reaches about 90%, using a pointed cell to scratch and scrape a proper number of scratches vertical to the transverse line of the bottom; cells were washed 3 times with PBS, and the scratched cells were removed and replaced with serum-free medium. Adding Lumbricus extract and negative control reagent (P BS) into the culture solution, respectively, adding 5% CO at 37 deg.C₂The cells were incubated under conditions, photographed under a microscope at 0, 12 and 24 hours, and the effect of earthworm extract treatment on cell migration and differentiation was counted using ImageJ.

FIG. 2 is a graph showing the scratch repair effect of different earthworm extracts on mouse NIH-3T3 fibroblasts, wherein a) is a curve of proliferation rate of mouse NIH-3T3 fibroblasts by different earthworm extracts, b) is the influence of different concentrations of earthworm extracts on cell activity, the earthworm extracts in the healing period (2 days after the operation of the mouse NIH-3T 3) are compared with the earthworm extracts without being cut in the healing period (2 days after the operation of the mouse NIH-3T 3) in the operation of the mouse NIH-3T3, only the earthworm extracts in the wound healing period (2 days after the operation of the mouse NIH-3T 3) have the obvious effect of promoting the scratch repair, and the medicinal effect of the earthworm extracts depends on the extract concentration, as shown in b) in FIG. 2, under the concentration of 1.6 mu L/per hole (500ug/ul), the activity of the earthworm extracts in the healing period of 2 days after the wound healing period has the highest effect of promoting the growth of the mouse fibroblasts, and the effect of inhibiting the growth of the earthworm extracts in the mouse NIH-3T3 fibroblasts is gradually improved as the optimal concentration of the mouse NIH-3T L μ.

After the earthworm extract with 2 days of body breaking is found to have the effect of obviously improving the mouse cell scar repairing effect, the effect of the earthworm extract with body breaking is better than that of the earthworm without body breaking for further verification. The mouse NIH-3T3 fibroblasts were scratched according to the procedure as described above, PBS, the unbroken earthworm extract and the broken body earthworm extract for 2 days were added, the width of the scratch was observed just after scratching, 12 hours and 24 hours after breaking, and the cell mobility was calculated to represent the wound repair rate.

The results are shown in FIG. 3, which is a graph of the scratch repair effect of different drugs on mouse NIH-3T3 fibroblasts at different times; wherein, a) is a microscopic observation image of scratch width, and b) is cell mobility; it can be seen that the earthworm extract in the period of body fracture healing has higher mouse wound repair promoting effect in 12 hours and 24 hours, and is particularly obvious in 24 hours.

Example 3 in vitro therapeutic effects of different earthworm extracts on wound repair in mice

After the earthworm extract with 2 days of body breaking is found to have the effect of obviously improving the mouse cell scar repairing on the mouse NIH-3T3 fibroblast scar repairing, the earthworm extract with body breaking effect superior to that of the earthworm without body breaking is further confirmed on a mouse animal model.

A punching wound model with the diameter of 0.5mm is made on the back of a BA L B/c mouse aged 12 weeks, 20 mu L of 0.02 mol/L of Phosphate Buffer Solution (PBS) with the pH value of 7.4 is dripped on a wound of a control group, 20 mu L of earthworm extract liquid for 2 days and earthworm extract liquid for a fracture are respectively dripped on a wound of an experimental group, observation and shooting are carried out every 0-10 days after the wound, the influence of drug treatment on wound repair is observed, wound tissues just after punching (0 day), 1 st, 3 rd, 6 th and 10 th days are taken for paraffin section and HE staining, and the wound healing and granulation growth conditions of the mouse are observed by a microscope.

FIG. 4 is a graph showing the effect of different drugs on the repair of skin defects in mice at different times; wherein a) is a microscopic view of the extent of the skin defect, b) is the area of wound healing; it can be seen that the wound area of the back of the mouse is reduced more rapidly and the repair promoting effect is more obvious compared with PBS and the earthworm extract without broken body in the broken and cured period, and the specific ratio can be shown as b) in FIG. 4.

As shown in fig. 5, HE staining of wound tissues of mice shows that the wound of mice treated with the earthworm extract in the 2-day-old-healed-period-broken body had a higher macrophage activation rate, the wound was significantly contracted in 6-10 days, the repair was faster, and the two groups were significantly different from PBS and the earthworm extract group without broken body. The earthworm extract in the fracture healing period can shorten the inflammation period and promote the healing of the skin wound of the mouse.

Example 4 transcriptomics and Mass Spectrometry studies of fragmented earthworm extracts

Extracting total RNA of the earthworm without breakage and the earthworm in the healing period (2 days after breakage) by using a Trizol method, sequencing by using a second-generation transcriptome, simultaneously carrying out mass spectrum detection and analysis on the extracts of the earthworm without breakage and the earthworm in the healing period, carrying out combined analysis of the transcriptome and the mass spectrum to find specific differential expression protein in the intersected earthworm extract in the healing period, analyzing the protein after NIH-B L AST comparison, and designing a primer clone target expression fragment.

As shown in fig. 6, which is a transcriptomic up-and down-regulation gene difference volcano plot of 2-day-disrupted and non-disrupted earthworms; analysis shows that 2624 genes of the earthworm with 2 days broken body are down-regulated and 2689 genes of the earthworm with 2 days broken body are up-regulated.

As shown in fig. 7, the transcriptomics KEGG functional enrichment analysis of earthworms with two days of disruption and earthworms without disruption shows the semantics of the characteristics of five gene products: in Environmental Information Processing (EIP), metabolism (M), Organic Systems (OS), Cellular Processes (CP) and Human Diseases (HD), KEGG functional analysis shows that most of the up-regulated genes are enriched in extracellular stroma, protein absorption and degradation, focal adhesion of cells and other pathways.

As shown in fig. 8, in the two-day-broken earthworm extract mass spectrum (AEE2D), the unbroken earthworm extract mass spectrum (EE) and the transcriptomics key enrichment gene wain map, mass spectrometry was performed on the 2-day-broken earthworm extract and the unbroken earthworm extract, and after combined analysis with the up-regulated gene in the transcriptome, it was found that the protein/gene was present in the 2-day-broken earthworm extract and was also up-regulated at the transcriptome level. Through Blast comparison analysis with an NIH database, the protein is found to be earthworm collagen (the amino acid sequence is shown as SEQ ID NO:1, and the nucleotide sequence is shown as SEQ ID NO: 2).

As shown in fig. 9, the mass spectrum (specific peptide segment RNGYK) of the collagen of the decapitated earthworm is shown.

Example 5 Synthesis of collagen

Selecting a conservative sequence fragment (shown as SEQ ID NO: 5) with the molecular weight of about 38 kD. according to a gene sequence of collagen obtained in transcriptomics, then selecting a proper restriction enzyme cutting site based on a cDNA sequence for coding the fragment, adding the restriction enzyme cutting site to the 5' end (which can be designed by a Primer Premier 5.0 and selects two restriction enzyme cutting sites of EcoR I and Xho I) of a PCR Primer (the sequences are shown as SEQ ID NO:3 and SEQ ID NO: 4), digesting and recovering an amplification fragment and a pET-32a plasmid by using two restriction enzymes of EcoR I and Xho I, connecting and transforming Escherichia coli DH5 α, selecting a positive bacterial plaque, and amplifying a recombinant plasmid.

After the recombinant plasmid is transferred into an expression strain B L21, positive bacterial plaque is selected to carry out induction for 3h under the condition that target protein is less than 37 ℃ and 1mMIPTG, the strain centrifugally recovered after induction is resuspended by using a binding buffer solution, and can be divided into two parts of sediment and supernatant after ultrasonic crushing and refrigerated centrifugation, the two parts of protein are subjected to SDS-PAGE electrophoresis, and observed after dyeing and decoloring, if the protein is more in the sediment, the sediment part is used for purification, and on the contrary, if a large amount of target protein exists in the supernatant, the supernatant part is purified, and the target protein in the supernatant part is more by observation.

The binding buffer containing the recombinant protein was applied to the column, and then the protein was eluted with 70mM imidazole, and the effluent was collected to obtain purified collagen.

Example 6 cellular experiments of collagen on wound repair in mice

2ug/ml of the collagen protein prepared in example 5 was subjected to the pharmacological test on NIH-3T3 according to the method described in example 2.

As shown in fig. 10, which is a microscopic image of the effects of collagen and PBS on the repair and promotion of mouse NIH-3T3 fibroblast scratches at 0 hour and 12 hours, it was found that the scratches after collagen treatment are narrower than the scratches of the cells treated by PBS of the control group at 12 hours, and it was confirmed that the collagen obtained by the separation method of the present invention indeed has a significant effect on promoting the healing of wounds.

It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

SEQUENCE LISTING

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gcatggactg gaggttctgg aaggtcagga agcaatggag caaccggaat ccaaggcccc 360

atgggaagaa ctggttccac tggaccgatt ggatcaactg gagccactgg cgcaactggc 420

gtgggtaata ctggagccac tggtcccgct ggtggacctg gagcaactgg tcttcaagga 480

aatacaggac caatgggaag aacaggcgac tggaatcgga agaacgggtt caacaggtgc 540

aactggacca attggagcta cgggaggttc tggactgcca ggaagcaatg gagcaaccgg 600

aatccaaggc cccatgggaa gaactggttc cactggaccg attggatcaa ctggagccac 660

tggcgcaact ggcgtgggta a 681

Claims

1. An isolated collagen protein, wherein said collagen protein is selected from the group consisting of:

(a) 1, a collagen having an amino acid sequence of SEQ ID NO; or

2. An isolated collagen gene, wherein the polynucleotide sequence of the collagen gene comprises a nucleotide sequence encoding the collagen of claim 1.

3. The gene of claim 2, wherein the sequence of the polynucleotide is shown in SEQ ID NO. 2.

4. A recombinant vector comprising the polynucleotide of claim 2 or 3.

5. A host cell comprising the recombinant vector of claim 4.

6. A method for producing the collagen according to claim 1, comprising: 1) culturing the host cell of claim 5; 2) isolating the collagen of claim 1 from the culture.

7. Use of the collagen of claim 1 for the preparation of a medicament for wound healing.

8. An antibody capable of specifically binding to the collagen according to claim 1.

9. A composition comprising the collagen of claim 1, the recombinant vector of claim 4, the host cell of claim 5, or the antibody of claim 8.