CN113444167B - Recombinant human collagen polypeptide and application thereof - Google Patents
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Abstract
The invention discloses a recombinant human collagen polypeptide and application thereof. The amino acid sequence of the recombinant human collagen polypeptide is shown as SEQ ID NO.2 or the amino acid sequence with at least 90 percent of identity with the SEQ ID NO. 2. The recombinant human collagen polypeptide can be used for inhibiting the growth of vascular endothelial cells, promoting cell proliferation and promoting cell adhesion.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a recombinant human collagen polypeptide and application thereof.
Background
Collagen is one of the most abundant proteins in the animal body, widely distributed in the skin, bone, tendon, ligament, cartilage and blood vessel of mammals, and is the main protein component of connective tissue and also an important constituent component of extracellular matrix. The collagen content in human body is 25% -30% of total protein, and it is rich in amino acids of glycine, proline and hydroxyproline. Collagen, a structural protein, plays an important role in protecting the body and supporting organs. This makes collagen have wide application in medicine, food, beauty and biological material, and has been the focus of research in recent years.
Collagen is widely present in the extracellular matrix and has the structure of a triple helix formed by winding three polypeptide chains. The types of collagen vary depending on the combination of the three alpha chains. To date, 26 genetically distinct collagen types have been discovered. They can be roughly classified into the following categories: fibril forming collagen, basement membrane collagen, microfibril collagen, anchor fibrils, transmembrane region collagen, incompletely characterized collagen. The main types are: type I collagen, the triple helix of which is mainly composed of alpha 1 and alpha 2 chains, has the highest content in bones, tendons, skin, cornea and ligaments; type II collagen, the triple helix component of which is [ alpha 1(II)] 2 Present in tissues such as hyaline cartilage, vitreous and intervertebral disc; type III collagen consisting of a triple helix of [ alpha 1(III)] 3 Mainly distributed in embryo, gastrointestinal tract and periodontal membrane; type IV collagen, the triple helix constituent form of which is [ alpha 1(IV)] 3 ，[α1(IV)] 2 α2(VI)，[α2(I)] 3 The composition of basement membrane has high hydroxylysine content and high sugar content, and contains both 4-hydroxyproline and 3-hydroxyproline.
Collagen is a scaffold for basement membrane, bone, cartilage and other structures, and can be used for treating burn, wound and corneal diseases. Collagen plays an important role in wound healing, organ development, tissue repair, etc., and clinical applications include: medical collagen injection for cosmetic and plastic, collagen membrane for treating burn and wound, collagen hemostatic sponge for stopping bleeding of wound, etc. The collagen is an important component of the skin extracellular matrix, and has the effects of moisturizing, supplementing skin collagen, resisting aging and the like. Because the collagen has the biological functions, the collagen has wide application in the medical and health fields (such as hemostasis, cosmetology, drug delivery, wound repair, drug sustained release and the like).
At present, the collagen is mainly obtained by extracting from animal skin, bone, and other tissues by adopting an acid hydrolysis method, an alkaline hydrolysis method and an enzyme hydrolysis method. Due to its origin in animal tissue, there is a risk of cross-infection for diseases of animal origin or infectious diseases of humans. With the increasing maturity of genetic engineering technology, the tendency of extracting collagen from animal tissues is gradually replaced by preparing recombinant collagen by adopting a molecular biological method. The recombinant collagen produced in a prokaryotic or eukaryotic expression system has better cell adhesion, histocompatibility, skin permeability and stability, can promote cell formation and growth, and has unique superiority (no virus hidden danger, water solubility, low rejection reaction and the like) compared with the traditional animal collagen.
Disclosure of Invention
The invention aims to provide a recombinant human collagen polypeptide which can be used for inhibiting a vascular growth factor, regulating the plasticity of endothelial cells, preparing a cell growth promoter and preparing a cell adhesion promoter. Can be widely applied to the fields of biochemical engineering and cosmetics.
The specific technical scheme of the invention is as follows:
1. a recombinant human collagen polypeptide, the amino acid sequence of which is shown in SEQ ID NO.2 or is an amino acid sequence with at least 90% identity with SEQ ID NO. 2.
2. A nucleic acid molecule encoding the recombinant human collagen polypeptide of claim 1, preferably wherein the nucleic acid molecule is tagged with a tag, further preferably wherein the tag is 6 His, GST, MBP or SUMO.
3. A vector comprising the nucleic acid molecule of claim 2, preferably wherein the vector is pPIC9k, pPICZ α A, pGAPZA, pHIL-S1 or pYAM 75P.
4. A host cell comprising the vector of item 3.
5. The host cell according to item 4, wherein the host cell is a yeast host cell, preferably Pichia pastoris, more preferably Pichia pastoris.
6. A method of preparing the recombinant human collagen polypeptide of claim 1, comprising the steps of:
the host cell according to claim 5 or 6, which is expressed and then isolated and purified.
7. The method according to item 6, wherein the separation and purification method is Ni-Ni affinity chromatography, reduced glutathione affinity chromatography, maltose affinity chromatography or SUMO affinity chromatography
8. Use of the recombinant human collagen polypeptide of item 1 or the recombinant human collagen polypeptide encoded by the nucleic acid molecule of item 2 or 3 or the recombinant human collagen polypeptide expressed by the vector of item 4 or 5 or the recombinant human collagen polypeptide produced by the host cell of item 6 or 7 in biochemical or cosmetic applications.
9. An agent that inhibits the growth of vascular endothelial cells or a pro-cell proliferation agent or a pro-cell adhesion agent comprising the recombinant human collagen polypeptide of item 1 or the recombinant human collagen polypeptide encoded by the nucleic acid molecule of item 2 or 3 or the recombinant human collagen polypeptide expressed by the vector of item 4 or 5 or the recombinant human collagen polypeptide produced by the host cell of item 6 or 7.
10. Use of the recombinant human collagen polypeptide of item 1 or the recombinant human collagen polypeptide encoded by the nucleic acid molecule of item 2 or 3 or the recombinant human collagen polypeptide expressed by the vector of item 4 or 5 or the recombinant human collagen polypeptide produced by the host cell of item 6 or 7 for the preparation of an agent that inhibits the growth of vascular endothelial cells, a pro-cell proliferative agent or a pro-cell adhesion agent.
ADVANTAGEOUS EFFECTS OF INVENTION
The recombinant human collagen polypeptide can be used for inhibiting the growth of vascular endothelial cells, promoting cell proliferation and promoting cell adhesion.
Drawings
FIG. 1 is a schematic diagram of a Pichia pastoris expression vector and a recombinant human collagen polypeptide expression vector.
FIG. 2 is a schematic diagram of the expression and purification of the obtained recombinant collagen, wherein M is protein Marker, 1 is recombinant protein expression, 2 is empty vector expression, 3 is flow-through, 4 is NTA-10mM impurity washing, 5 is NTA-40mM impurity washing, 6 is NTA-200mM elution, and 7 is protein after ultrafiltration concentration.
FIG. 3-1 is a schematic diagram of the inhibitory effect of the recombinant human collagen polypeptide of the present invention and commercially available collagen on vascular endothelial cells.
FIG. 3-2 is a schematic diagram showing the inhibition rate of the recombinant human collagen polypeptide of the present invention on vascular endothelial cells.
FIG. 4-1 is a schematic diagram of the cell proliferation promoting effect of the recombinant human collagen polypeptide of the present invention and commercially available collagen.
FIG. 4-2 is a schematic representation of the cell proliferation promotion of the recombinant human collagen polypeptides of the invention.
FIG. 5-1 is a schematic diagram of the cell adhesion promotion effect of the recombinant human collagen polypeptide of the present invention and commercially available collagen.
FIG. 5-2 is a schematic representation of the cell adhesion promotion effect of the recombinant human collagen polypeptides of the present invention.
Detailed Description
The present invention is described in detail in the following description of embodiments with reference to the figures, in which like numbers represent like features throughout the figures. While specific embodiments of the invention are shown in the drawings, 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, it is to be understood that the description is for the purpose of illustration 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 human collagen polypeptide, the amino acid sequence of which is shown in SEQ ID NO.2 or is an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity with SEQ ID NO. 2.
The recombinant collagen polypeptide is creatively designed by the inventor on the basis of collagen, and can be used for inhibiting the growth of vascular endothelial cells and promoting the proliferation and adhesion of cells, wherein,
the amino acid sequence shown in SEQ ID NO.2 is as follows:
IKGDRGEIGPPGPRGEDGPEGPKGRGGPNGDPGPLGPPGEKGKLGVP GLPGYPGRQGPKGSIGFPGFPGANGEKGGRGTPGKPGPRGQRGPTGPRGE RGPRGITGKPGPKGNSGGDGPAGPPGERGPGEFYFDLRLKGDK
the invention provides a nucleic acid molecule, wherein the nucleic acid molecule codes the recombinant human collagen polypeptide.
The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1, and the nucleotide sequence is as follows:
CGGAATTCATCAAGGGTGATCGGGGGGAGATCGGCCCACCCGGT CCCAGGGGAGAAGATGGCCCTGAAGGCCCAAAGGGTCGCGGAGGTCC CAATGGTGACCCCGGTCCTCTGGGACCCCCTGGGGAGAAGGGAAAAC TCGGAGTCCCAGGGTTACCAGGGTATCCAGGAAGACAAGGACCAAAG GGCTCTATTGGATTCCCTGGATTTCCTGGCGCCAATGGAGAGAAGGGC GGCAGGGGGACCCCTGGAAAGCCAGGACCGCGGGGGCAGCGAGGCC CAACGGGTCCGAGGGGTGAAAGAGGCCCCCGGGGCATCACTGGGAA GCCTGGCCCCAAGGGCAACTCCGGAGGTGACGGCCCAGCTGGCCCTC CTGGTGAACGGGGACCCGGTGAGTTTTATTTCGACTTGCGGCTCAAAG GTGACAAAGCGGCCGCAA
the nucleic acid molecules may include those comprising naturally and/or non-naturally occurring nucleotides and bases, for example those having backbone modifications, which refers to polymers of nucleotides, such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA and PNA. Nucleotide sequence refers to the linear sequence that constitutes a nucleic acid molecule.
In some cases, the nucleic acid molecule comprises cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs of the invention, e.g., for codon optimization. In some cases, the sequences may be designed to contain terminal restriction site sequences for cloning into a vector.
In some cases, the nucleic acid molecule encoding the recombinant human collagen polypeptide 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 human collagen polypeptide is codon optimized. In general, codon optimization involves balancing the percentage of codons selected with the abundance of human transfer RNA disclosed, such that none is overloaded or restricted. 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 that are balanced with human frequency of use. Typically, the redundancy of amino acid codons is such that different codons encode one amino acid. In some embodiments, in selecting a codon for a substitution, it may be desirable that the resulting mutation be a silent mutation such that the codon change does not affect the amino acid sequence. In general, the last nucleotide of a codon can remain unchanged without affecting the amino acid sequence.
In one embodiment, the nucleic acid molecule is tagged with a tag, preferably 6 × His, GST (glutathione mercaptotransferase), MBP (maltose binding protein) or SUMO (small ubiquitin related modifier).
The present invention provides a vector comprising the nucleic acid molecule as described above.
In one embodiment, the vector is pPIC9k, pPICZ α A, pGAPZA, pHIL-S1 or pYAM 75P.
For example, one or more nucleic acids encoding the recombinant human collagen polypeptides 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) which are specific for the type of host (e.g., bacterial, fungal, plant or animal) into which the vector is to be introduced, 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 a recombinant human collagen polypeptide, the nucleic acid encoding the recombinant human collagen polypeptide 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. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny 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, by introducing the vector into host cells using an electric shock or using CaCl 2 The method is to introduce exogenous DNA into prokaryotic cell or introduce exogenous DNA into eukaryotic cell by liposome mediated transfection method or transfer gene into host cell by virus vector infection method.
In one embodiment, the host cell is a yeast host cell, preferably pichia, further preferably pichia pastoris, e.g. as GS115, KM71 strain.
The invention provides a method for preparing the recombinant human collagen polypeptide, which comprises the following steps:
the host cell is expressed and then separated and purified to obtain the recombinant vector.
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 examples of the culture conditions include YPD, BMGY, BMMY, and YNB.
The expression mode is not limited in any way, and the expression can be confirmed as required, for example, the expression is induced expression, and the inducer for the induced expression can be IPTG, beta-galactoside, methanol, ethanol and the like, preferably methanol.
The method of separation and purification is not limited in any way, and may be determined according to the method, and for example, Ni-nickel affinity chromatography, reduced glutathione affinity chromatography, maltose affinity chromatography or SUMO affinity chromatography may be used.
The recombinant human collagen polypeptide provided by the invention can be used for inhibiting the growth of vascular endothelial cells and promoting the proliferation and adhesion of cells.
The recombinant collagen polypeptide provided by the invention is an amino acid sequence obtained by design on the basis of collagen, and the recombinant collagen polypeptide with the amino acid sequence shown in SEQ ID NO.2 can be used for inhibiting the growth of vascular endothelial cells and promoting the proliferation and adhesion of cells.
The invention provides application of the recombinant human collagen polypeptide in biochemical engineering or cosmetics.
The invention provides an active agent for inhibiting the growth of vascular endothelial cells, which comprises the recombinant human collagen polypeptide or the recombinant human collagen polypeptide coded by the nucleic acid molecule or the recombinant human collagen polypeptide expressed by the vector or the recombinant human collagen polypeptide produced by the host cell.
The invention provides a cell growth promoter, which comprises the recombinant human collagen polypeptide or the recombinant human collagen polypeptide coded by the nucleic acid molecule or the recombinant human collagen polypeptide expressed by the carrier or the recombinant human collagen polypeptide produced by the host cell.
The invention provides a cell adhesion promoter, which comprises the recombinant human collagen polypeptide or the recombinant human collagen polypeptide coded by the nucleic acid molecule, or the recombinant human collagen polypeptide expressed by the carrier or the recombinant human collagen polypeptide produced by the host cell.
The recombinant human collagen polypeptide or the recombinant human collagen polypeptide coded by the nucleic acid molecule or the recombinant human collagen polypeptide expressed by the vector or the recombinant human collagen polypeptide produced by the host cell is applied to the preparation of an active agent for inhibiting the growth of vascular endothelial cells, a cell proliferation promoting agent or a cell adhesion promoting agent.
Examples
The invention is described generically and/or specifically with respect to 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 used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1 preparation of recombinant human collagen polypeptide
1. Construction of recombinant human collagen polypeptide expression vector
The nucleotide sequence (the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2) of the recombinant collagen peptide synthesized by the corporation of Weitusheng bioengineering (Shanghai). And designing an upstream primer and a downstream primer containing enzyme cutting sites by using the nucleotide sequence fragment as a template to perform PCR reaction. Wherein, the upstream primer is added with 6 His label to facilitate the subsequent purification.
The sequence of the upstream primer P1 is shown as SEQ ID NO. 3, and the nucleotide sequence is as follows: 5'-CGGAATTCATCAAGGGTGATCG-3', which contains an EcoRI restriction endonuclease cut site;
the sequence of the downstream primer P2 is shown as SEQ ID NO. 4, and the nucleotide sequence is as follows: 5'-TTGCGGCCGCTTTGTCACCT-3', which contains a NotI restriction endonuclease cleavage site.
The reaction conditions of PCT are as follows: pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, 30 cycles, and extension at 72 ℃ for 10min, wherein the reaction system is shown in Table 1:
TABLE 1 PCR reaction System
| Reagent | Dosage of |
| Template DNA | 1μL |
| PrimeSTAR Max DNA Polymerase | 12.5μL |
| Primer P1 | 0.5μL |
| Primer P2 | 0.5μL |
| ddH 2 O | 0.5μL |
The recovered PCR product and the expression vector pPIC9K (purchased from Invitrogen) were digested with the restriction enzymes EcoRI and Not I. The enzyme digestion reaction system is shown in table 2:
TABLE 2 expression vectors and digestion of PCR products of collagen polypeptides
The enzyme digestion products were recovered with a gel recovery kit and ligated with T4 DNA ligase (purchased from Saimer Feishell science and technology (China) Co., Ltd.) at 16 ℃ for 30min, and the ligation reaction was as shown in Table 3:
TABLE 3 ligation of expression vectors to collagen polypeptides
| Reagent | Dosage of |
| Cleaved PCR product fragment (50 ng/. mu.L) | 2μL |
| Cleaved pPIC9K vector (25 ng/. mu.L) | 2μL |
| 10×Ligation Buffer | 1μL |
| T4 DNA Ligase | 0.5μL |
| ddH 2 O | 4.5μL |
Transforming the ligation product into Escherichia coli DH5 alpha competent cells (purchased from Saimer Feishell science and technology (China)) and mixing, ice-bathing for 30min at 42 ℃ for 60s (without shaking), and ice-bathing for 1-2 min; adding 500 mu L LB culture medium, and incubating for 1h at 37 ℃ by a shaking table; shaking the bacterial solution, coating 100 μ L of the solution on ampicillin agar plate (LB/AP +), and culturing at 37 deg.C for 12-16 hr. Single colonies were picked as templates on agar plates and PCR was performed with the upstream primer P1(SEQ ID NO:3) and the downstream primer P2(SEQ ID NO: 4). The positive clones screened by PCR were further identified by digestion with restriction enzymes EcoRI and NotI. Clones with both positive results were subjected to sequencing analysis. The sequencing primer is a universal sequencing primer aiming at the plasmid pPIC9K, if the DNA sequencing result is consistent with the designed gene sequence, the successful construction of the expression vector is determined, and meanwhile, the recombinant escherichia coli transformed into the empty vector pPIC9K is constructed as a control.
2. Expression and purification of recombinant human collagen polypeptides
Inoculating a transformant with correct DNA sequencing into an LB liquid culture medium containing 100mg/mL Amp, carrying out shaking culture at 37 ℃ and 220rpm overnight, carrying out bulk extraction on pPIC9k-COL-S plasmid (adopting a plasmid macroextraction kit purchased from Tiangen Biotechnology (Beijing) Co., Ltd.), taking a proper amount of the plasmid, carrying out gradient dilution, and carrying out agarose gel electrophoresis to detect the plasmid concentration. The vector was linearized by single cleavage of pPIC9k-COL-S with Sacl I, the cleavage system of which is shown in Table 4:
TABLE 4 linearized cleavage reaction of recombinant collagen polypeptides
After the above digested DNA was purified, 10. mu.L of linearized DNA pPIC9k-COL-S was added to 80. mu.L of GS115 yeast competent cells, the operation scheme of which is shown in FIG. 1, and transferred to a pre-cooled 0.2cm electrode cup and ice-cooled for 5 min. Carefully wiping the ice on the electrode cup, putting the electrode cup into an electrode groove, screwing the electrode cup, putting down a glass cover, adjusting the voltage to 1500V, adjusting the resistance to 200 omega, and adjusting the capacitance to 50 muF for conversion. Immediately after the shock was completed, the lid was opened and 1mL of ice-precooled 1M sorbitol was added, and after gentle mixing the whole liquid was transferred to a 1.5mL sterile centrifuge tube and coated with MD plates. And (3) inversely culturing the plate in a 30 ℃ biochemical incubator for 2-5 days until a single colony appears. Colony PCR positive screening was performed using COL-S primers (upstream primer P1 and downstream primer P2), and colonies growing the desired band were spotted in sequence on YPD plates containing G418 at 0.25mg/mL to 4.0mg/mL in a clean bench for high copy screening.
Inoculating the high-copy single colony in a YPD culture medium for 30 ℃, carrying out shaking culture at 220rpm for overnight, taking a proper amount of culture solution to be transferred to a BMGY culture medium for 30 ℃, carrying out shaking culture at 220rpm for overnight till OD600 reaches 2-8, taking a proper amount of culture to be transferred to a 50mL sterilized centrifuge tube, centrifuging at 4000rpm at room temperature for 3min, then discarding supernatant, resuspending the thallus in the BMMY culture medium until OD600 is equal to 1, carrying out induction culture at 30 ℃, carrying out induction culture at 250rpm, and carrying out induction culture on a yeast strain GS115 and the yeast strain GS115 transformed with the empty vector pPIC9k by the same method. Add 250. mu.L of methanol every 24 h. 1mL of the sample was sampled every 24 hours, centrifuged at 12000rpm for 1min, and then the supernatant and the cells were stored at-20 ℃ for subsequent detection. After 5 days of induction, the culture was stopped, and the fermentation broth was centrifuged to store the supernatant and the cells, respectively.
And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection on the fermentation supernatant, wherein the concentration of the concentrated gel is 5%, the concentration of the separation gel is 15%, and the sample amount of the supernatant is 15 mu L. And (3) running the gel for 30min under the voltage of 60V, adjusting the voltage to 100V, continuing electrophoresis for about 1-2 h until bromophenol blue reaches the bottom of the gel plate, stopping electrophoresis, dyeing with Coomassie brilliant blue R-250 for 30min, decoloring with decoloring solution for about 1h, and replacing the decoloring solution for several times until protein bands are clearly shown. The results showed that the empty vector pPIC9k was not expressed in yeast strain protein, whereas pPIC9k-COL-S collagen polypeptide was expressed in yeast strain GS115, as shown in FIG. 2.
The recombinant protein utilizes Ni-nickel affinity chromatography, the fermented supernatant is added into a Ni-nickel affinity chromatography medium and mixed, so that the target protein is fully combined with Ni-nickel affinity chromatography glue, and the combination flow rate is 0.6 mL/min. Washing the base line by NTA-10mM imidazole buffer solution at the flow rate of 1 mL/min; then washing the hybrid protein by using NTA-40mM imidazole buffer solution at the flow rate of 1mL/min, and eluting the target protein by using NTA-200mM imidazole buffer solution at the flow rate of 1 mL/min; the target protein was found in the supernatant and in the recovery after NTA-200mM elution, and the size was about 14KD, which was consistent with the expected results. After the purified protein is subjected to ultrafiltration and centrifugation, the sample is loaded with 20 mu g, and the protein is substantially free of impurity bands and has the purity of over 95 percent, as shown in figure 2.
Experimental example 1 application of recombinant human collagen polypeptide in inhibition of vascular endothelial cells
The recombinant human collagen polypeptide described in example 1 was applied to VEC (human vascular endothelial cells) cells to perform a cytostatic experiment. The method comprises the following specific steps: human HUVEC cells (purchased from ATCC cell bank, catalog number: CRL-1730) were seeded into sterile 96-well plates with 200. mu.l of culture medium per well, and M199 complete medium was used for cell culture, to which 0.25mg/mL, 0.5mg/mL, 1mg/mL, and 2mg/mL of recombinant human (COL-S) collagen polypeptide was added per well, respectively. Three multiple wells are set for each group concentration, and an equal volume of culture solution is added into a blank control group, and the blank control group is placed in an incubator at 37 ℃ for 48 hours. Discarding the supernatant, adding 100. mu.L of culture solution and 50. mu.L of MTT solution to each well, shaking, placing in an incubator, incubating for 4h, after 4h, aspirating the mixture, adding 150. mu.L of DMSO to dissolve purple formazan crystal, shaking for 10min, placing in an microplate reader, detecting the wavelength at 570nm, measuring the absorbance value of each well, and determining the HUVEC endothelial cell inhibition data of the recombinant human collagen polypeptide as shown in Table 5, which is shown in 3-1 together with the inhibition effect of commercial collagen (purchased from Abcam company) on vascular endothelial cells, and the growth inhibition curve as shown in 3-2, wherein,
inhibition rate (blank OD-experimental OD) × 100%/blank.
TABLE 5 Absorbance values for inhibition of vascular endothelial cells by recombinant human collagen polypeptides at various concentrations and commercially available collagen
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|
| Control | 1.036 | 0.912 | 0.986 |
| 0.25mg/mL | 0.735 | 0.769 | 0.786 |
| 0.5mg/mL | 0.617 | 0.623 | 0.653 |
| 1mg/mL | 0.396 | 0.312 | 0.306 |
| 2mg/mL | 0.469 | 0.475 | 0.482 |
| Commercial collagen 1mg/mL | 0.607 | 0.615 | 0.649 |
As can be seen from Table 5 and FIGS. 3-1 to 3-2, the inhibition rate of the recombinant collagen of the present invention on vascular endothelium is above 60%, while the inhibition rate of the commercially available collagen is below 40%, which indicates that the recombinant collagen of the present invention has a strong inhibition effect on vascular endothelial cells.
Experimental example 2 application of recombinant human collagen polypeptide in promoting cell proliferation
Cell proliferation experiments were performed using the recombinant human collagen polypeptides described in example 1, acting on HSF (human skin fibroblasts) cells. The method comprises the following specific steps: human HSF cells (purchased from ATCC cell bank, catalog number HTX2132) were seeded into sterile 96-well plates in 200. mu.L of culture medium per well, and DMEM/F12 complete medium was used for cell culture, and recombinant human (COL-S) collagen polypeptides were added at 0.25mg/mL, 0.5mg/mL, 1mg/mL, and 2mg/mL, respectively, per well. Three multiple wells are set for each group concentration, and an equal volume of culture solution is added into a blank control group, and the blank control group is placed in an incubator at 37 ℃ for culturing for 48 hours. Discarding the supernatant, adding 100. mu.L of culture solution and 50. mu.L of MTT solution into each well, shaking, incubating for 4h, sucking out the mixture after 4h, adding 150. mu.L of DMSO to dissolve purple formazan crystal, shaking for 10min, placing into a microplate reader, detecting the wavelength at 570nm, measuring the absorbance value of each well, and comparing the data of HSF (human skin fibroblast) cell proliferation promoted by recombinant collagen polypeptide with data shown in Table 6, with a schematic diagram of human skin fibroblast proliferation with commercially available collagen (purchased from Abcam company) shown in 4-1 and a graph of proliferation rate shown in 4-2,
proliferation rate (experimental OD-blank OD) × 100%/blank.
TABLE 6 Absorbance values for proliferation of skin fibroblasts by recombinant collagen polypeptides at various concentrations and by commercially available collagen
| |
|
|
|
| Control of | 0.523 | 0.562 | 0.539 |
| 0.25mg/mL | 0.625 | 0.692 | 0.612 |
| 0.5mg/mL | 0.723 | 0.713 | 0.701 |
| 1mg/mL | 0.953 | 0.975 | 0.964 |
| 2mg/mL | 0.946 | 0.956 | 0.976 |
| Commercially available collagen (1mg/mL) | 0.715 | 0.736 | 0.729 |
As can be seen from Table 6 and FIGS. 4-1 to 4-2, the cell proliferation promoting rate of the recombinant collagen of the present invention is close to 80%, while the cell proliferation promoting rate of the commercially available collagen is lower than 40%, which indicates that the recombinant collagen of the present invention can significantly promote the proliferation of cells.
Experimental example 3 application of recombinant human collagen polypeptide in promoting cell adhesion
The recombinant human collagen polypeptide described in example 1 was used to perform a cell adhesion promotion experiment on HSF (human skin fibroblast) cells. The method comprises the following specific steps: inoculating human HSF cells into a sterile 96-well plate, adding 200 mu L of culture medium into each well, adding PBS only into a blank control group, placing for 2h at 37 ℃, washing each group for 3 times by using PBS, washing off unadhered cells, adding 100 mu L of culture solution and 50 mu L of MTT solution into each well, shaking up, placing in an incubator for incubation for 4h, then sucking out a mixed solution, adding 150 mu L of DMSO to dissolve purple crystal formazan, shaking for 10min, placing in an enzyme reader, detecting the wavelength of 570nm, and measuring the absorbance value of each well.
The data of the collagen polypeptide promoting HSF (human skin fibroblast) cell adhesion are shown in Table 7, the schematic adhesion effect of the collagen polypeptide and the commercial collagen (purchased from Abcam company) on human skin fibroblast is shown in 5-1, the adhesion rate curve is shown in 5-2, wherein,
the cell adhesion promoting rate was ═ (experimental OD-blank OD) × 100%/blank.
TABLE 7 absorbance values for adhesion promotion of skin fibroblasts by recombinant collagen polypeptides at various concentrations and by commercially available collagen
| |
|
|
|
| Control | 0.326 | 0.352 | 0.348 |
| 0.25mg/mL | 0.432 | 0.415 | 0.426 |
| 0.5mg/mL | 0.510 | 0.521 | 0.513 |
| 1mg/mL | 0.629 | 0.611 | 0.634 |
| 2mg/mL | 0.613 | 0.602 | 0.615 |
| Commercial collagen (1mg/mL) | 0.523 | 0.551 | 0.534 |
As can be seen from Table 7 and FIGS. 5-1 to 5-2, the recombinant collagen of the present invention has a cell adhesion promoting rate of 80%, whereas the commercially available collagen has a cell adhesion promoting rate of less than 60%, indicating that the recombinant collagen of the present invention can significantly promote cell adhesion.
In conclusion, the recombinant human collagen polypeptide provided by the invention can inhibit the growth of vascular endothelial cells and promote the proliferation and adhesion of cells, so that the recombinant human collagen polypeptide can be applied to the fields of biochemical engineering or cosmetics.
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 made to the above embodiments according to the technical essence of the present invention, without departing from the technical solution of the present invention, still belong to the protection scope of the technical solution of the present invention.
Sequence listing
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<120> recombinant human collagen polypeptide and application thereof
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Claims (15)
1. The amino acid sequence of the recombinant human collagen polypeptide is shown as SEQ ID NO. 2.
2. A nucleic acid molecule, wherein said nucleic acid molecule encodes the recombinant human collagen polypeptide of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is labeled with a label.
4. The nucleic acid molecule of claim 3, wherein the tag is 6 His, GST, MBP or SUMO.
5. A vector, wherein the vector comprises the nucleic acid molecule of any one of claims 2-4.
6. The vector of claim 5, wherein the vector is pPIC9k, pPICZ α A, pGAPZA, pHIL-S1, or pYAM 75P.
7. A host cell comprising the vector of claim 5.
8. The host cell of claim 7, wherein the host cell is a yeast host cell.
9. The host cell of claim 7, wherein the host cell is Pichia pastoris.
10. The host cell of claim 7, wherein the host cell is Pichia pastoris.
11. A method of preparing the recombinant human collagen polypeptide of claim 1, comprising the steps of:
the host cell of any one of claims 7-10, which is expressed and then isolated and purified.
12. The method of claim 11, wherein the separation and purification method is Ni-nickel affinity chromatography, reduced glutathione affinity chromatography, maltose affinity chromatography or SUMO affinity chromatography.
13. Use of the recombinant human collagen polypeptide of claim 1 or encoded by the nucleic acid molecule of any one of claims 2 to 4 or expressed by the vector of claim 5 or 6 or produced by the host cell of any one of claims 7 to 10 for the preparation of a biochemical or cosmetic product.
14. An agent that inhibits the growth of vascular endothelial cells or a pro-cell proliferation agent or a pro-cell adhesion agent comprising the recombinant human collagen polypeptide of claim 1 or encoded by the nucleic acid molecule of any one of claims 2-4 or expressed by the vector of claim 5 or 6 or produced by the host cell of any one of claims 7-10.
15. Use of the recombinant human collagen polypeptide of claim 1 or encoded by the nucleic acid molecule of any one of claims 2 to 4 or expressed by the vector of claim 5 or 6 or produced by the host cell of any one of claims 7 to 10 for the preparation of an agent that inhibits the growth of vascular endothelial cells, a pro-cell proliferation agent or a pro-cell adhesion agent.
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| CN113444167B (en) * | 2021-07-15 | 2022-09-30 | 陕西巨子生物技术有限公司 | Recombinant human collagen polypeptide and application thereof |
| CN113735966B (en) * | 2021-09-29 | 2022-11-01 | 陕西巨子生物技术有限公司 | Anti-tumor recombinant collagen and preparation method and application thereof |
| CN114316030B (en) * | 2022-01-27 | 2023-06-30 | 西安巨子生物基因技术股份有限公司 | Transdermal absorptive type I recombinant collagen and application thereof |
| CN116478274B (en) * | 2022-07-19 | 2023-12-15 | 山西锦波生物医药股份有限公司 | Preparation method of biological synthetic human body structural material |
| CN117304306A (en) * | 2023-09-28 | 2023-12-29 | 广州普言生物科技有限公司 | Recombinant III type collagen Pro.C3 and preparation method and application thereof |
| CN117003857B (en) * | 2023-09-28 | 2024-01-05 | 英特菲尔(成都)生物制品有限责任公司 | Collagen with transdermal absorption performance and preparation method and application thereof |
| CN117285616B (en) * | 2023-10-07 | 2024-02-27 | 广东省卓肽医药有限公司 | Recombinant humanized I+III type collagen and application thereof |
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| WO2023284900A2 (en) | 2023-01-19 |
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