CN116813749A - Recombinant humanized III type collagen and preparation method and application thereof - Google Patents
Recombinant humanized III type collagen and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The application relates to the technical field of biology, and particularly discloses a recombinant humanized III-type collagen and a preparation method and application thereof. The nucleotide sequence of the recombinant humanized III type collagen is shown as (a) or (b): the nucleotide sequence of (a) is shown as SEQ ID NO. 5; (b) A protein derived from (a) wherein the nucleotide sequence in (a) is substituted, deleted or added with one or more amino acids and has collagen activity. The recombinant humanized III type collagen designed by the application has better stability and activity, and the recombinant humanized III type collagen can obviously promote the migration of fibroblasts and presents certain dose dependence.
Description
Technical Field
The application relates to the technical field of biology, in particular to recombinant humanized III type collagen, a preparation method and application thereof.
Background
Collagen has good biocompatibility, low immunogenicity, biodegradability and good mechanical properties, and the collagen is widely applied to biomedical and biomedical materials at present; collagen-based biomaterials have been currently used in skin tissue repair, soft tissue fillers, bone tissue regeneration fillers, surgical hemostatic materials, tissue fillers; artificial blood vessels, etc.
There are 28 types of collagen, and different types of collagen have been found to have different functions. The most content of the human body is type I collagen, which accounts for more than 85 percent, and the next is type III collagen, which accounts for 10 to 15 percent. Type I collagen is high in bone, skin, tendons and cornea, and type III exists in blood vessels and neogenesis skin tissue. Type I and type III collagens are the major components of the extracellular matrix responsible for the integrity of tissue structures.
Type III collagen molecules, which consist of three alpha 1 chains, are a protein encoded by the COL3A1 gene in humans, and have a long triple-helical domain. Type III collagen forms smaller fibers, has lower tensile strength, and is present in movable organs and periodically stretched tissues. Type III collagen is the main structural component of hollow organs, such as the large blood vessels, uterus and intestinal tract. Type III collagen provides tensile strength and integrity to many organs, and studies have shown that type III collagen has several other important functions. Other functions of type III collagen include interactions with platelets in the blood coagulation cascade, which is also an important signaling molecule for wound healing. In tissues where type III collagen fibrils have a smaller diameter than type I collagen fibrils, type I and type III collagen occur in the same fibrils, in this case, type III collagen can regulate the diameter of the fibrils.
Collagen used at present mainly has two sources, namely, collagen extracted from animal tissues and recombinant DNA technology for preparing recombinant collagen. Collagen extracted from animal tissues may have an intact collagen molecular structure, and present some immunogenicity, but also present a risk of introducing viruses. The collagen prepared by the recombinant DNA technology has better biocompatibility, water solubility and stability, and does not have the risk of introducing viruses. More importantly, the collagen prepared by the tissue extraction mode is mainly type I collagen, and the type III collagen can be prepared by a recombinant DNA technology.
Because of the specific amino acid composition of the collagen, how to improve the stability and the expression level of the collagen and whether the collagen can form a correct folding structure prepared by a recombinant DNA technology is a key aspect for restricting the application of the collagen. Therefore, in the preparation process of the collagen through the genetic engineering manner, a proper collagen domain needs to be selected, and the amino acid sequence of the selected collagen domain is reasonably optimized, so that the preparation efficiency of the collagen can be improved, and the stability and activity of the prepared collagen can be improved.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide a recombinant humanized III-type collagen and a preparation method and application thereof. The recombinant humanized III type collagen has better stability and cell migration promoting activity, and can obviously promote the migration of fibroblasts.
In a first aspect, the present application provides a recombinant humanized type III collagen having a nucleotide sequence as set forth in (a) or (b):
(a) The nucleotide sequence is shown as SEQ ID NO. 5;
(b) A protein derived from (a) wherein the nucleotide sequence in (a) is substituted, deleted or added with one or more amino acids and has collagen activity.
In the technical scheme of the application, the nucleotide sequence of human III collagen is taken as a reference (NM_ 000090.3), a specific receptor binding domain on the III collagen and on the cell surface is selected, the stability of peptide fragments in the candidate region is analyzed, and finally, the upper peptide fragment (the amino acid sequence is shown as SEQ ID NO.1 or the nucleotide sequence is shown as SEQ ID NO. 2) of the human III collagen is screened out, namely, a recombinant humanized III collagen basic structural unit (GERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGE NGAP); based on the basic structural unit of the recombinant humanized III type collagen, the recombinant humanized III type collagen is RC4, consists of 228 amino acids, and has the amino acid sequence shown in SEQ ID NO.3 or the nucleotide sequence shown in SEQ ID NO. 4.
The nucleotide sequence of the recombinant humanized III type collagen RC4 is optimized by combining codon preference and a translation suspension technology, and the DNA sequence of the recombinant humanized III type collagen RC4 is synthesized through total genes, wherein the nucleotide sequence is shown as SEQ ID NO. 5.
The recombinant humanized III type collagen is efficiently and soluble expressed in escherichia coli; high purity collagen can be prepared by affinity chromatography, which can significantly promote migration of fibroblasts.
In a second aspect, the present application provides a nucleic acid molecule encoding said recombinant humanized type III collagen.
In a third aspect, the present application provides an expression vector carrying the nucleic acid molecule.
In a fourth aspect, the present application provides a host cell comprising said recombinant humanized type III collagen or said nucleic acid molecule or said expression vector.
As a preferred embodiment of the host cell of the present application, the host cell uses pET-28a as an expression vector.
As a preferred embodiment of the host cell according to the application, the host cell is an E.coli BL21 (DE 3) competent cell.
The fifth object of the present application is to provide a method for preparing the recombinant humanized type III collagen, comprising the steps of:
and expressing the recombinant humanized III type collagen by using the host cell, and then separating and purifying to obtain the recombinant humanized III type collagen.
In some specific embodiments, the use of host cell expression refers to culturing the host cell, the culture medium and culture conditions being well known to those skilled in the art. The expression pattern is not limited in any way, and it can be confirmed as needed, for example, the expression pattern is induced expression. The separation and purification method comprises salting out method, ultrafiltration method, affinity chromatography, gel filtration chromatography, acid-base precipitation method and membrane separation method. Preferably, the separation and purification method is an affinity chromatography method.
In a sixth aspect, the present application provides a cell migration promoting agent comprising the recombinant humanized type III collagen.
The seventh object is to provide the application of the recombinant humanized III type collagen in preparing a reagent for promoting cell migration.
Preferably, the concentration of the recombinant humanized type III collagen in the cell migration promoting agent is 12.5-50 mug/mL. The recombinant humanized III type collagen can obviously promote migration of fibroblasts, and presents certain dose dependence.
In an eighth aspect, the present application provides the use of said recombinant humanized type III collagen, or said recombinant humanized type III collagen encoded by said nucleic acid molecule, or recombinant humanized type III collagen produced by said host cell, in the preparation of a food, cosmetic or medical device product.
Compared with the prior art, the application has the following beneficial effects:
the recombinant humanized III type collagen designed by the application has better stability and activity, experiments show that the recombinant humanized III type collagen can promote the mobility of fibroblasts to be 24.62% and 31.41% under the conditions of concentration of 12.5 mug/mL and 50 mug/mL, and compared with a control group, the recombinant humanized III type collagen can obviously promote the mobility of the fibroblasts and presents certain dose dependence. The recombinant humanized III type collagen designed by the application is efficiently expressed in a soluble form in escherichia coli by optimizing the nucleotide sequence, and can be prepared into high-purity collagen by affinity chromatography.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of recombinant humanized type III collagen RC4 (Panel 1: protein standard, panel 2: uninduced expressed bacteria, panel 3: IPTG-induced expressed bacteria, panel 4: thallus disruption supernatant, panel 5: thallus disruption centrifugation pellet, panel 6: ni-NTA affinity chromatography penetration peak, panel 7: ni-NTA affinity chromatography 60mM imidazole elution, and Panel 8: ni-NTA affinity chromatography 250mM imidazole elution);
FIG. 2 is a diagram of an imaging of a cell migration promoting cell of recombinant humanized type III collagen RC 4;
FIG. 3 is a graph showing the results of cell migration promotion by recombinant humanized type III collagen RC4.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
The techniques and procedures described or referenced herein are conventional methods generally known and often used by those skilled in the art.
The main materials involved in the examples are as follows: host bacterium E.coli BL21 (DE 3) (Merck), plasmid pET-28a (Merck), pre-dye protein Marker were purchased from Thermo Fisher Scientific company, ni Sepharose TM 6Fast Flow was purchased from GE company, and CCK-8 kit was purchased from Japan homozygote company. The other reagents were all analytically pure reagents.
NTA-20 buffer (20 mmol/L Tris-HCl, pH 8.0+0.15mol/L NaCl+20mmol/L imidazole), NTA-60 buffer (20 mmol/L Tris-HCl, pH 8.0+0.15mol/L NaCl+60mmol/L imidazole), NTA-250 buffer (20 mmol/L Tris-HCl, pH 8.0+0.15mol/L NaCl+250mmol/L imidazole).
The application provides recombinant humanized III type collagen, and the nucleotide sequence of the recombinant humanized III type collagen is shown as SEQ ID NO. 5.
The application also provides a nucleic acid molecule for encoding the recombinant humanized III type collagen,
in the above embodiments, the nucleic acid molecule is a generic name of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biomacromolecule compounds polymerized from a number of nucleotide monomers, and are one of the most basic substances for life. Nucleotide sequence refers to the order of bases in DNA or RNA. The nucleic acid molecules comprise cDNA and in some cases, the nucleic acid molecules may be modified for use in the vectors of the application, such as for codon optimization. In some cases, the sequences may be designed to contain terminal restriction site sequences for cloning into a vector. The nucleic acid molecule may 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.
The application also provides an expression vector carrying the nucleic acid molecule.
In a specific embodiment, an expression vector refers to a DNA molecule capable of self-replication in genetically engineered recombinant DNA techniques to transfer a DNA fragment (gene of interest) to a recipient cell. As the most commonly used, simplest vectors in genetic engineering, three parts must be included: a genetic marker gene, a replication region, and a gene of interest. In addition to the commonly used E.coli plasmid vectors, many other artificially constructed plasmid vectors suitable for use in microorganisms, yeasts, plants, etc. have been developed. Vectors include, but are not limited to: a single-stranded, double-stranded or partially double-stranded nucleic acid molecule; nucleic acid molecules comprising one or more free ends, without free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments may be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing expression of a gene of interest. Such vectors are referred to herein as "expression vectors". The recombinant expression vector may comprise a form suitable for expressing the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements, which may be selected based on the host cell for expression, which may be operably linked to the nucleic acid sequence to be expressed.
The application also provides a host cell for expressing the recombinant humanized III type collagen.
In a particular embodiment, a host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present application. "host cell" encompasses any progeny of a parent cell that is not exactly identical to the parent cell due to mutations that occur during replication. The host cell may be any cell useful in the production of recombinant humanized type III collagen of the application. To produce recombinant humanized type III collagen, nucleic acid encoding recombinant humanized type III 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 specifically binding to genes encoding recombinant collagen). The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be identical in nucleic acid content to the parent cell, but may contain mutations. Methods for introducing vectors into host cells are well known, for example, using electrotransformation to introduce vectors into host cells, and may also be transfection, microinjection techniques, gene gun techniques, liposome-mediated methods, and the like. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from E.coli.
Example 1 design of recombinant humanized type III collagen
In this example, the nucleotide sequence of human type III collagen is used as a reference (NM_ 000090.3), the binding domain of the cell surface integrin receptor on type III collagen is selected, and the stability of the peptide fragment in the candidate region is analyzed by collagen stability analysis software, and finally the peptide fragment on type III collagen (the amino acid sequence is shown as SEQ ID NO.1 or the nucleotide sequence is shown as SEQ ID NO. 2) is selected, namely, the basic structural unit of recombinant humanized type III collagen (GERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGE NGAP);
SEQ ID NO.1：
GERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGEN GAP；
SEQ ID NO.2：
GGCGAGCGTGGCTTACCGGGCCCACCGGGCATTAAAGGTCCGGCGGGCATTC
CGGGCTTTCCGGGCATGAAAGGCCATCGCGGCTTTGATGGCCGCAACGGCGA
AAAGGGCGAAACCGGTGCCCCGGGCTTAAAGGGTGAAAATGGTCTGCCGGGTGAGAATGGCGCGCCG；
based on the basic structural unit of the recombinant humanized III type collagen, the recombinant humanized III type collagen is RC4, consists of 228 amino acids, and has the amino acid sequence shown in SEQ ID NO.3 or the nucleotide sequence shown in SEQ ID NO. 4.
The nucleotide sequence of the recombinant humanized III type collagen RC4 is optimized by combining with the codon preference of escherichia coli and through a translation suspension technology, the DNA sequence of the recombinant humanized III type collagen RC4 is synthesized through total genes, the synthesized DNA sequence is cloned into a pET-28a plasmid, and the obtained nucleotide sequence of the recombinant humanized III type collagen is shown as SEQ ID NO. 5.
SEQ ID NO.3：GERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPPGAPGPCCGG；
SEQ ID NO.4：
GGCGAGCGTGGCTTACCGGGCCCACCGGGCATTAAAGGTCCGGCGGGCATTC
CGGGCTTTCCGGGCATGAAAGGCCATCGCGGCTTTGATGGCCGCAACGGCGA
AAAGGGCGAAACCGGTGCCCCGGGCTTAAAGGGTGAAAATGGTCTGCCGGGT
GAGAATGGCGCGCCGGGCGAGCGTGGCTTACCGGGCCCACCGGGCATTAAAG
GTCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCTTTGAT
GGCCGCAACGGCGAAAAGGGCGAAACCGGTGCCCCGGGCTTAAAGGGTGAA
AATGGTCTGCCGGGTGAGAATGGCGCGCCGGGCGAGCGTGGCTTACCGGGCC
CACCGGGCATTAAAGGTCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGG
CCATCGCGGCTTTGATGGCCGCAACGGCGAAAAGGGCGAAACCGGTGCCCCG
GGCTTAAAGGGTGAAAATGGTCTGCCGGGTGAGAATGGCGCGCCGGGCGAGC
GTGGCTTACCGGGCCCACCGGGCATTAAAGGTCCGGCGGGCATTCCGGGCTTT
CCGGGCATGAAAGGCCATCGCGGCTTTGATGGCCGCAACGGCGAAAAGGGCG
AAACCGGTGCCCCGGGCTTAAAGGGTGAAAATGGTCTGCCGGGTGAGAATGGCGCGCCGGGACCTCCTGGTGCCCCTGGTCCTTGCTGTGGTGGTTAA；SEQ ID NO.5：
GGCGAACGCGGCTTACCGGGCCCACCGGGCATTAAAGGTCCGGCGGGTATTC
CGGGCTTCCCGGGCATGAAAGGCCACCGTGGTTTTGACGGCCGCAATGGCGA
GAAAGGCGAAACCGGCGCGCCGGGTCTGAAAGGTGAGAACGGCTTACCGGG
CGAGAACGGCGCCCCGGGCGAACGTGGCCTGCCGGGCCCACCGGGCATCAAG
GGTCCGGCGGGCATCCCGGGTTTCCCGGGCATGAAGGGCCATCGCGGCTTTG
ATGGCCGCAACGGCGAAAAAGGCGAGACGGGTGCGCCGGGTCTGAAGGGCG
AAAACGGCCTGCCGGGTGAAAACGGCGCGCCGGGCGAACGCGGCTTACCGG
GTCCGCCGGGCATTAAAGGCCCGGCGGGTATTCCGGGCTTCCCGGGCATGAA
AGGCCATCGTGGTTTTGACGGTCGCAACGGTGAGAAAGGCGAGACCGGCGCG
CCGGGTTTAAAGGGCGAAAATGGCCTGCCGGGCGAGAATGGTGCGCCGGGCG
AACGCGGCTTACCGGGCCCGCCGGGTATCAAAGGTCCGGCCGGCATCCCGGG
CTTTCCGGGCATGAAAGGTCACCGTGGTTTTGATGGCCGTAACGGCGAAAAA
GGCGAAACCGGCGCGCCGGGCCTGAAGGGTGAAAACGGTTTACCGGGCGAAAATGGCGCGCCGGGCCCACCGGGTGCGCCGGGTCCGTGCTGCGGCGGCTAA。
Example 2 preparation method of recombinant humanized type III collagen
(1) Preparation of recombinant humanized type III collagen RC 4-expressing strains:
(1) preparation of competent cells of E.coli BL21 (DE 3): the preparation process is detailed in the third edition of the molecular cloning experiment guide; [ Mei ] J. Cym Brookfield, huang Peitang translation.
(2) The expression vector pET-28a-RC4 (cloning of the DNA sequence of recombinant humanized type III collagen RC4 into pET-28a plasmid) was transformed into E.coli BL21 (DE 3) competent cells: the transformation process is detailed in the third edition of the molecular cloning experiment guide; [ Mei ] J. Cym Brookfield, huang Peitang translation.
(2) Recombinant humanized type III collagen RC4 induced expression and solubility analysis:
inoculating the expression strain pET-28a-RC4 obtained in the step (1) into 30mL of LB medium containing 50 mug/mL kanamycin, culturing at 37 ℃ and 180rpm, and obtaining the OD 600 When=0.8, IPTG was added at a final concentration of 1mm, induced to express at 37 ℃ for 4 hours, and then centrifuged at 5000g at 4 ℃ for 10min to collect the cells. The cells were resuspended in 20mmol/L Tris-HCl (pH 8.0,0.15mol/L NaCl) buffer, homogenized and disrupted at high pressure (800 bar), centrifuged at 18000g at 4℃for 30min, and the supernatant and pellet were respectively subjected to subsequent SDS-PAGE (5% gel, 12% gel) and Western blot analysis.
(3) Recombinant humanized III type collagen RC4 shake flask fermentation and purification:
and (3) inoculating the recombinant humanized III type collagen RC4 expression strain obtained in the step (1) into 1L of LB culture medium with the kanamycin content of 50 mug/mL respectively, and carrying out shake flask fermentation and induction according to the expression conditions. Centrifuging at 4 ℃ for 6000 Xg for 10min to collect thalli, and precipitating the thalli according to the volume ratio of 1:10 ratio resuspended in NTA-20 buffer, and cells were broken up homogeneously at high pressure (1000 bar). Supernatant was collected by centrifugation at 25000 Xg for 30min at 4 ℃.
The bacterial cell disruption and centrifugation supernatant is purified by Ni-NTA affinity chromatography, and the specific steps are as follows: loading the supernatant onto a Ni-NTA affinity chromatographic column with a bed volume of 20mL, washing the supernatant back to a base line with NTA-20 buffer solution at a flow rate of 1mL/min, washing the impurity protein with NTA-60 buffer solution at a flow rate of 2mL/min, and eluting the target protein with NTA-250 buffer solution; the purified recombinant humanized III collagen RC4 is subjected to Sephadex G-25 molecular sieve deimidazole (shown in figure 1). In fig. 1: band 1 represents a protein molecular weight standard, band 2 represents an engineering bacterium which does not induce expression, band 3 represents an engineering bacterium which induces expression, band 4 represents a cell disruption centrifugal supernatant, band 5 represents a cell disruption centrifugal precipitation, band 6 represents a protein purification penetration peak sample, band 7 represents a protein purification hybrid protein, and band 8 represents a protein-purified protein of interest RC4.
Example 3 evaluation of recombinant humanized type III collagen RC4 cell migration promoting Activity
1. BALB/3T3 cells in the logarithmic growth phase were taken, the cells were digested with 0.25% trypsin, resuspended in DMEM serum-free medium, and counted.
2. 200. Mu.L of the cells were collected and the cell density was 1.5X10 5 the/mL cells were added to the upper chamber of the Transwell culture chamber, and recombinant humanized III-type collagen RC4 (obtained in example 2) was added to the upper chamber of the Transwell at different concentrations, with the final concentrations of recombinant humanized III-type collagen RC4 being 12.5. Mu.g/mL and 50. Mu.g/mL, respectively. 600. Mu.L of medium containing 5% serum was added to the lower chamber of a Transwell culture chamber, and a 24-well culture plate was placed at 37℃in 5% CO 2 Is cultured in an incubator.
3. The cell culture plate was removed, excess fluid in the upper chamber was aspirated, and washed 2 times with PBS. Cells in the Transwell upper chamber were scraped with a cotton swab, and the chamber was placed in a chamber containing 600. Mu.L of 4% paraformaldehyde and fixed at room temperature for 20 minutes.
4. The Transwell chamber was washed 3 times with PBS, the PBS was rubbed off the chamber with a cotton swab, and the Transwell chamber was placed in 0.2% crystal violet and stained at room temperature for 20 minutes. After the staining was completed, the chamber was washed 3 times with PBS, and the cotton swab was gently rotated in the upper chamber to suck the water.
5. A glass slide is placed on a positive microscope, and the cell chamber is turned upside down for photographing and observation. The biometric analysis was performed using Image J "Analyze Particles".
The imaging diagram of the recombinant humanized III type collagen RC4 cell migration promoting cell is shown in FIG. 2, and the result of the cell migration promoting of the recombinant humanized III type collagen RC4 is shown in FIG. 3.
Transwell experiment results show that: the mobility of the recombinant humanized III type collagen RC4 for promoting the fibroblasts under the conditions of the concentration of 12.5 mug/mL and 50 mug/mL is 24.62 percent and 31.41 percent respectively, and compared with a control group, the recombinant humanized III type collagen RC4 can obviously promote the migration of the fibroblasts and presents certain dose dependence.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.
Claims (10)
1. A recombinant humanized type III collagen, wherein the nucleotide sequence of the recombinant humanized type III collagen is as set forth in (a) or (b):
(a) The nucleotide sequence is shown as SEQ ID NO. 5;
(b) A protein derived from (a) wherein the nucleotide sequence in (a) is substituted, deleted or added with one or more amino acids and has collagen activity.
2. A nucleic acid molecule encoding the recombinant humanized type III collagen of claim 1.
3. An expression vector carrying the nucleic acid molecule of claim 2.
4. A host cell comprising the recombinant humanized type III collagen of claim 1 or the nucleic acid molecule of claim 2 or the expression vector of claim 3.
5. The host cell of claim 4, wherein the host cell comprises pET-28a as an expression vector.
6. The host cell of claim 4, wherein the host cell is an E.coli BL21 (DE 3) competent cell.
7. A method of preparing recombinant humanized type III collagen according to claim 1, comprising the steps of:
expressing the recombinant humanized type III collagen by using the host cell according to any one of claims 4 to 6, and then separating and purifying the recombinant humanized type III collagen.
8. A cell migration promoting agent comprising the recombinant humanized type III collagen according to claim 1.
9. Use of recombinant humanized type III collagen according to claim 1 for the preparation of a reagent for promoting cell migration.
10. Use of the recombinant humanized type III collagen according to claim 1, or the recombinant humanized type III collagen encoded by the nucleic acid molecule according to claim 2, or the recombinant humanized type III collagen produced by the host cell according to any one of claims 4-6, for the preparation of a food, cosmetic or medical device product.
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