CN117247942A - Human collagen gene, production method and detection method of collagen - Google Patents
Human collagen gene, production method and detection method of collagen Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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, cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
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- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The application discloses a gene of human collagen, a production method and a detection method of the collagen, and belongs to the field of bioengineering. The gene sequence has the sequence shown in SEQ ID: the nucleotide sequence shown in NO. 1. The production method of the human collagen is characterized by comprising the following steps: constructing escherichia coli genetic engineering bacteria; fermenting and culturing escherichia coli genetic engineering bacteria; inducing and expressing recombinant human collagen; purifying recombinant human collagen; the beneficial effects of the application are that a new collagen sequence is provided; and the escherichia coli expression system is adopted, so that the large-scale amplification is suitable, one-round fermentation can be completed within 20 hours, the production cost is very low, and the sequence in the gene is subjected to codon optimization aiming at the escherichia coli expression system, so that the yield is further improved.
Description
Technical Field
The application relates to the field of bioengineering, in particular to a gene of human collagen, a production method and a detection method of the collagen.
Background
The humanized collagen has weak antigenicity, is favorable for cell adhesion, can induce cell proliferation and differentiation, can provide a support for cell ingrowth collagen deposition and neovascularization, and can provide necessary amino acid for wound repair by degradation products, so that the humanized collagen becomes an important material for preparing application fields such as wound dressing, cosmetics, tissue engineering, medical appliances and the like.
Collagen molecules form a special supercoiled structure consisting of three polypeptide chains that are formed by intra-chain hydrogen bonds and are supported only by inter-chain hydrogen bonds. The helical structure is a left-handed helix with a basic repeat of 3 amino acid residues, typically Gly-X-Pro. Gly is necessary for the formation of hydrogen bonds in collagen, and has no side chains, so that collagen can be closely packed. At a higher structural level, the collagen supercoils are further associated into collagen fibrils. In organisms, collagen synthesis and modification begin from procollagen, undergo various chemical changes such as hydroxylation, glycosylation, cross-linking and the like, and are subjected to complex regulation and control of various biological enzymes. Procollagen contains globular heads and tails in addition to collagen chains. Without these heads and tails, the collagen chains do not fold into the correct triple helix, thus lacking the biological activity of collagen. Thus, collagen prepared according to the original gene sequence cannot spontaneously form a correct spatial structure in vitro. Such difficulties have severely hampered the development and production of human collagen;
the traditional method for producing collagen is to treat animal-derived tissues by acid, alkali and enzymolysis methods to extract collagen derivatives. The collagen extracted by the methods has lost the original biological activity and cannot be applied to the biomedical field to play a real function. Along with the development of modern biotechnology, people continuously try to prepare recombinant human collagen in animal, plant and microorganism expression systems by utilizing transgenic technology, and various defects of the traditional extraction process are overcome. Foreign research institutions cultivate mice containing human collagen genes to obtain human collagen-containing milk, but the production cost is too high, the production period is too long, and large-scale production cannot be put into.
Disclosure of Invention
The content of the present application is intended to introduce concepts in a simplified form that are further described below in the detailed description. The section of this application is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
In order to solve the technical problems mentioned in the background section above, a first object of the present application is to provide a gene of human collagen, wherein the gene sequence has the sequence shown in seq id no: the nucleotide sequence shown in NO. 1.
A second object of the present application provides a method for producing a source collagen, comprising the steps of: constructing escherichia coli genetic engineering bacteria; fermenting and culturing escherichia coli genetic engineering bacteria; inducing and expressing recombinant human collagen; purification of recombinant human collagen.
Further, the method for constructing the escherichia coli genetic engineering bacteria comprises the following steps: optimally selecting DNA fragments of human III type collagen genes, and synthesizing recombinant plasmids; transferring the recombinant gene into an escherichia coli expression strain, and screening to obtain escherichia coli genetic engineering bacteria.
Further, the method for fermenting and culturing the escherichia coli genetic engineering bacteria comprises the following steps: adding 0.1%50 mug/mL kana antibiotic into LB liquid culture medium, picking single colony, mixing uniformly, and culturing in a shaking table at 37 ℃ and 220rpm for 12h; adding 0.1%50 μg/mL kana antibiotic into LB liquid culture medium, adding 1.0% of overnight culture bacterial liquid, placing in a shaking table at 37deg.C and 220rpm for culturing, adding inducer IPTG for induction after OD600 value is 0.4-0.6 when logarithmic growth phase is measured, and inducing for 24h at low temperature of 16deg.C and 220 rpm.
Further, the method for purifying recombinant human collagen comprises the following steps: resuspending the bacteria with Tris buffer, sonicating, centrifuging and collecting supernatant; and purifying the supernatant by using a nickel ion affinity column to obtain the recombinant human collagen.
The third object of the present application is to provide a method for detecting human collagen, comprising the following steps:
carrying out MTT experiments on the humanized collagen to obtain MTT experiment results; performing a cell scratch experiment on the humanized collagen to obtain a cell scratch experiment result; cell adhesion measurement and relative adhesion calculation were performed on human collagen to obtain calculation results.
Further, the method for carrying out MTT experiment on the human collagen and obtaining MTT experiment result comprises the following steps:
BALB/3T3 cells were incubated at 37℃with 5% CO 2 The cells were cultured in a cell incubator, and the cell density and status were observed under an inverted microscope every day. Cell passage or cell seeding is performed when the cells grow to 80-90% of the flask. Cells were grown at 3X 10 using complete medium 3 Planting the seeds/holes in a 96-well plate, setting four compound holes at 100 mu L of each hole, culturing for 24 hours at 37 ℃ under the condition of 5% carbon dioxide, and then changing the culture solution into a maintenance culture solution for starvation treatment for 24 hours; the maintenance culture solution was discarded, and 100. Mu.L of recombinant collagen prepared in the maintenance medium was added thereto at a final concentration of 0mg/mL, 0.01mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, respectively. The cells were cultured at 37℃under 5% carbon dioxide for 72 hours. The blank control group is only added with a maintenance medium; 10. Mu.L of MTT solution was added to each well, and the mixture was incubated at 37℃for 4 hours under 5% carbon dioxide. Carefully sucking and discarding the supernatant in the holes, adding 100 mu LDMSO into each hole, fully blowing and uniformly mixing, then placing into an enzyme labeling instrument, measuring the light absorption value at the wavelength of 570nm, and obtaining the MTT experiment result.
Further, the method for carrying out cell scratch experiments on the human collagen and obtaining the cell scratch experiment result comprises the following steps:
marking three lines longitudinally for each well behind the 6-well plate with a marker pen. About 10X 10 per well 5 And (3) a plurality of cells. The goal is to reach a confluence state of 95-100% after 24 hours of culture; after cell culture for 24h, the well plate was vertically aligned with a 10. Mu.L gun head to form scratches by pushing down the longitudinal streaks, the cells were rinsed 3 times with D-PBS, the scraped cells were removed, and 2mL of recombinant collagen prepared in maintenance medium was added to 6 wells at final concentrations of 0mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, respectively. The blank control group is only added with a maintenance medium; at 37 ℃,5% CO 2 Culturing in an incubator, taking the intersection point of transverse and longitudinal scribing lines as a core after 0h and 24h, taking pictures under a 40-time microscope, and obtaining photos of 9 parts per hole; measuring the area of the scratch area, and calculating the mobility of each group of cells by dividing the total area of the transitional cells of the fixed scratch area by the initial area of the fixed scratch area; and obtaining the cell scratch experiment result.
Further, the method for performing cell adhesion measurement and relative adhesion calculation on the human collagen comprises the following steps: 100. Mu.L of recombinant collagen samples were added to each 96-well plate; incubate for 4h at 37℃in a 5% CO2 incubator. Excess D-PBS coating solution was removed from the wells, 100. Mu.L of 0.5% BSA-PBS solution was added, and incubated in a 5% CO2 incubator at 37℃for 1h. After removing the liquid in the wells, washing three times with D-PBS, discarding the cleaning solution, sealing with a sealing film, and standing at 4 ℃ for later use. Before use, placing the cell plate in an incubator for temperature return; BALB/3T3 cells were cultured at 37℃in a 5% CO2 cell incubator, and the cell density and status were observed under an inverted microscope every day. Cell passage or cell seeding is performed when the cells grow to 80-90% of the flask. Cells were grown at 5X 10 using complete medium that had been pre-mixed with Hoechst33342 fluorescent stain 4 Each well was seeded in a 96-well plate with 100. Mu.L of each well, three duplicate wells were set, covered with tin foil and incubated at 37℃with 5% CO 2. Incubation time is 1h; fluorescent photographing was performed on each sample well before centrifugation. After photographing, each well was filled with 250 μ L D-PBS to form a "reverse bendThe lunar surface "was covered with a sealing film, placed upside down, centrifuged at 250 centrifugal force for 5min, the sealing film was discarded and the supernatant was removed from the well. After washing 1 time with D-PBS, 100. Mu. L D-PBS was added, and each well was photographed; and calculating and obtaining a calculation result.
The beneficial effects of this application lie in:
1. the utility model provides a novel collagen sequence;
2. the escherichia coli expression system is suitable for large-scale amplification, one-round fermentation can be completed within 20 hours, the production cost is very low, and the sequence in the gene is codon optimized for the escherichia coli expression system, so that the yield is further improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, are included to provide a further understanding of the application and to provide a further understanding of the application with regard to the other features, objects and advantages of the application. The drawings of the illustrative embodiments of the present application and their descriptions are for the purpose of illustrating the present application and are not to be construed as unduly limiting the present application.
In addition, the same or similar reference numerals denote the same or similar elements throughout the drawings. It should be understood that the figures are schematic and that elements and components are not necessarily drawn to scale.
In the drawings:
FIG. 1 is an electrophoresis chart after purification, ultrafiltration and concentration, wherein an M lane is a marker, and a 1 lane is a protein electrophoresis band;
FIG. 2 is a graph showing the results of a test for the cell proliferation promoting capacity of recombinant collagen;
FIG. 3 is a representative photograph of scratch areas of 0h and 24h corresponding to different concentrations of recombinant collagen;
FIG. 4 is a graph showing the effect of recombinant collagen on cell mobility;
FIG. 5 is a representative photograph of different concentrations of recombinant collagen before and after centrifugation;
FIG. 6 shows the relative adhesion results of recombinant collagen at different concentrations to cells.
Detailed Description
Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While certain embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure 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. It should be understood that the drawings and embodiments of the present disclosure are for illustration purposes only and are not intended to limit the scope of the present disclosure.
It should be noted that, for convenience of description, only the portions related to the present utility model are shown in the drawings. Embodiments of the present disclosure and features of embodiments may be combined with each other without conflict.
It should be noted that the terms "first," "second," and the like in this disclosure are merely used to distinguish between different devices, modules, or units and are not used to define an order or interdependence of functions performed by the devices, modules, or units.
It should be noted that references to "one", "a plurality" and "a plurality" in this disclosure are intended to be illustrative rather than limiting, and those of ordinary skill in the art will appreciate that "one or more" is intended to be understood as "one or more" unless the context clearly indicates otherwise.
The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
With reference to figures 1-6 of the drawings,
natural collagen is insoluble in water, has nonuniform properties, is difficult to be used by a human body, and is often required to be treated by chemical means for use. Moreover, collagen products currently on the market are all obtained from animal tissues such as pigs, cows and fish, and it is difficult to avoid viral infection, and the collagen products are not compatible with human bodies, which can lead to immune rejection and allergic symptoms. If the extract is extracted from human placenta raw materials, the source is limited, and the method faces severe punishment of law. Therefore, the collagen can only be used in cosmetics and health care products, and the original biological functions of the collagen can not be exerted at all.
Therefore, the direction needs to be put into the natural collagen of the human body, and the structure of the natural collagen of the human body is very complex structurally, so that the human-derived collagen is extremely difficult to express and prepare in a large quantity by the conventional means. Therefore, the preparation of the human collagen has a great investment in research and development costs.
A gene of human collagen, which is characterized in that the gene sequence has the sequence table SEQ ID: the nucleotide sequence shown in NO. 1.
A method for producing human collagen comprises the following steps
Constructing escherichia coli genetic engineering bacteria;
fermenting and culturing escherichia coli genetic engineering bacteria;
inducing and expressing recombinant human collagen;
purification of recombinant human collagen.
Specifically, the method for constructing the escherichia coli genetic engineering bacteria comprises the following steps:
optimally selecting DNA fragments of human III type collagen genes, and synthesizing recombinant plasmids; transferring the recombinant gene into an escherichia coli expression strain, and screening to obtain escherichia coli genetic engineering bacteria.
Specifically, the method for fermenting and culturing the escherichia coli genetic engineering bacteria comprises the following steps:
adding 0.1%50 mug/mL kana antibiotics into LB liquid culture medium, putting escherichia coli genetic engineering bacteria into LB liquid culture medium, and picking single colony in LB culture medium; uniformly mixing the picked single colonies, and placing the single colonies in a shaking table at 220rpm at 37 ℃ for culturing for 12 hours to obtain a cultured bacterial liquid; adding 0.1%50 μg/mL kana antibiotic into LB liquid culture medium, adding 1.0% of cultured bacterial liquid, placing into a shaking table at 37 ℃ and 220rpm for culturing, adding inducer IPTG for induction after OD600 value is 0.4-0.6 when the culture enters logarithmic phase, wherein the induction condition is that the low temperature is 16 ℃ and the induction is 220rpm for 24 hours; centrifuging to collect thalli under the conditions of 13000rpm,1min and 4 ℃; the cells collected by centrifugation were either stored at-20℃or immediately subjected to further purification.
Specifically, the purification method of the recombinant human collagen comprises the following steps:
adding the centrifugally collected thalli into a buffer solution of BufferA, fully blowing and uniformly mixing, centrifuging at 13000rpm for 10min at 4 ℃, and reserving the sediment; adding a buffer solution of BufferA into the precipitate, re-suspending the precipitate to enable the precipitate to be fully dissolved in the solution, and continuously ultrasonically crushing for 3s at intervals of 7s each time by utilizing an ultrasonic crusher under the condition that the ultrasonic intensity is 80 percent until the solution is in a semitransparent state; centrifuging at 13000rpm and 4 ℃ for 30min, discarding the sediment-reserved supernatant solution to obtain an induction product.
The nickel ion affinity column (Ni-NTA) column was washed with PB buffer, then the column and Trx-III type collagen solution were mixed and incubated, gently shaken for 30min at room temperature or on ice, then the column was put on, and the hetero proteins and eluted proteins were washed with PB solution containing 10mM,20mM,30mM,500mM imidazole gradient. The resulting product was dialyzed overnight and concentrated; and purifying the supernatant by using a nickel ion affinity column to obtain the recombinant human collagen.
The detection method of the human collagen comprises the following steps:
carrying out MTT experiments on the humanized collagen to obtain MTT experiment results;
performing a cell scratch experiment on the humanized collagen to obtain a cell scratch experiment result;
cell adhesion measurement and relative adhesion calculation were performed on human collagen to obtain calculation results.
Specifically, the method for carrying out MTT experiment on the humanized collagen and obtaining MTT experiment result comprises the following steps:
1. cell culture: BALB/3T3 cells (mouse embryo fibroblasts) were cultured at 37℃with 5% CO 2 The cells were cultured in a cell incubator, and the cell density and status were observed under an inverted microscope every day. Cell passage or cell seeding is performed when the cells grow to 80-90% of the flask. Cells were grown at 3X 10 using complete medium 3 Planting the seeds/holes in a 96-well plate, setting four compound holes at 100 mu L of each hole, culturing for 24 hours at 37 ℃ under the condition of 5% carbon dioxide, and then changing the culture solution into a maintenance culture solution for starvation treatment for 24 hours;
2. the maintenance culture solution was discarded, and 100. Mu.L of recombinant collagen prepared in the maintenance medium was added thereto at a final concentration of 0mg/mL, 0.01mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, respectively. The cells were cultured at 37℃under 5% carbon dioxide for 72 hours. The blank control group is only added with a maintenance medium;
3. MTT test: 10. Mu.L of MTT solution was added to each well, and the mixture was incubated at 37℃for 4 hours under 5% carbon dioxide. Carefully sucking and discarding the supernatant (comprising a maintenance culture medium and an MTT solution) in each well, adding 100 mu LDMSO into each well, fully blowing and uniformly mixing, and then placing the mixture into an enzyme labeling instrument, and measuring the light absorption value at the wavelength of 570 nm;
4. obtaining an MTT experiment result; the structure is shown in FIG. 2, and as the concentration of recombinant collagen increases, the absorbance at 570nm continuously increases, indicating that the recombinant collagen has biological activity of promoting cell proliferation.
Specifically, the method for carrying out cell scratch experiments on the human collagen and obtaining the cell scratch experiment result comprises the following steps:
1. cell culture: marking three lines longitudinally on the back of the 6-hole plate by using a marker pen. About 10X 10 per well 5 And (3) a plurality of cells. The goal is to reach a confluent state of 95-100% after 24 hours of cultivation.
2. Scratch test: after cell culture for 24h, the well plate was vertically aligned with a 10. Mu.L gun head to form scratches by pushing down the longitudinal streaks, the cells were rinsed 3 times with D-PBS, the scraped cells were removed, and 2mL of recombinant collagen prepared in maintenance medium was added to 6 wells at final concentrations of 0mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, respectively. The blank group was added with maintenance medium only.
3. At 37 ℃,5% CO 2 Culturing in incubator, taking intersection point of transverse and longitudinal streaks as core after 0h and 24h, photographing under 40 times of microscope, and obtaining 9 photos of each hole.
4. The scratch area was measured and the cell mobility of each group was calculated by dividing the total area of the stationary scratch area transitional cells by the initial area of the stationary scratch area.
5. Obtaining a cell scratch experiment result; the results are shown in fig. 3 and 4, wherein fig. 3 is a representative photograph of 0h and 24h scratch areas corresponding to different concentrations of recombinant collagen, and it can be seen that the areas of the 0h scratch areas of different groups are close, but the areas of the 24h scratch areas gradually decrease with increasing concentrations of recombinant collagen, indicating that the cell migration ability is enhanced. FIG. 4 is a graph showing the effect of recombinant collagen on cell mobility. Compared with the control group (0 mg/mL of recombinant collagen), the mobility of the cells continuously increases for 24 hours along with the increase of the concentration of the recombinant collagen, and the recombinant collagen has the biological activity of promoting the migration of the cells.
Specifically, the method for measuring cell adhesion and calculating relative adhesion of the human collagen comprises the following steps:
1. and (3) preparing a coating: 100. Mu.L of recombinant collagen (0 mg/mL, 0.5mg/mL, 1mg/mL, 2 mg/mL) samples were added to the 96-well plates, respectively. 3 wells per sample coating were prepared and incubated for 4h at 37℃in a 5% CO2 incubator. Excess D-PBS coating solution was removed from the wells and 100. Mu.L of 0.5% BSA-PBS solution, 37℃and 5% CO was added 2 Incubate in incubator for 1h. After removing the liquid in the wells, washing three times with D-PBS, discarding the cleaning solution, sealing with a sealing film, and standing at 4 ℃ for later use. Before use, the cell plates are placed in an incubator for tempering.
2. Cell preparation: BALB/3T3 cells were cultured at 37℃in a 5% CO2 cell incubator, and the cell density and status were observed under an inverted microscope every day. Cell passage or cell seeding is performed when the cells grow to 80-90% of the flask. Using complete medium which had been pre-mixed with Hoechst33342 fluorescent stain (10%), cells were seeded at 5X 104 cells/well in 96-well plates, 100. Mu.L per well, three multiplexed wells were set, covered with tinfoil at 37℃with 5% CO 2 And (5) incubating. Incubation time was 1h.
3. And (3) detection: fluorescent photographing was performed on each sample well before centrifugation. After photographing, each well was filled with 250 μ L D-PBS to form a "reverse meniscus" and covered with a sealing membrane, placed upside down, centrifuged at 250 centrifugal force for 5min, the sealing membrane was discarded and the supernatant was removed from the well. After washing 1 time with D-PBS, 100. Mu. L D-PBS was added thereto, and each well was photographed.
(3 connected fields of view are photographed per well)
4. And (3) calculating results: the number of fluorescently labeled nuclei was counted using an automatic cell count procedure and the number of cells before and after centrifugation was determined. The percentage adhesion was calculated as follows:
v-percentage of adhesion;
nt-number of cells after centrifugation;
nc-number of cells before centrifugation.
The relative cell adhesion ratio was calculated as:
p-relative cell adhesion ratio;
v1, average value of adhesion percentage of each compound hole of the collagen sample;
v 2-average percent adhesion for each duplicate well of negative control.
5. Obtaining a calculation result; as can be seen from fig. 5 and 6, the recombinant collagen has a certain cell adhesion, wherein fig. 5 is a representative photograph of different concentrations of recombinant collagen before and after centrifugation; FIG. 6 shows the relative adhesion results of recombinant collagen at different concentrations to cells.
The functions of the human collagen are in the aspects of maintaining the extracellular environment, maintaining the normal physiological functions of tissues and organs, repairing the injury of the body, and the like. Collagen is a natural biological resource, has the biological tissue compatibility required by other high molecular materials, and has the supporting elasticity and the degradability for cells, so that the collagen can be widely applied to industries such as medicines, cosmetics and the like.
The foregoing description is only of the preferred embodiments of the present disclosure and description of the principles of the technology being employed. It will be appreciated by those skilled in the art that the scope of the utility model in the embodiments of the present disclosure is not limited to the specific combination of the above technical features, but encompasses other technical features formed by any combination of the above technical features or their equivalents without departing from the spirit of the utility model. Such as the above-described features, are mutually substituted with (but not limited to) the features having similar functions disclosed in the embodiments of the present disclosure.
Gene sequence table:
SEQID：NO.1
ATGGGCCACCATCATCACCACCACGGAGATGACGATGACAAAGGTCCGCGTGGCGAACGTGGTGAAGCAGGTATTCCGGGTGTTCCGGGTGCGAAGGGCGAGGATGGTAAAGACGGCTCCCCGGGTGAGCCGGGCGCGAACGGTCTGCCGGGCGCGGCAGGCGAGCGCGGTGCTCCGGGCTTCCGCGGTCCGGCGGGCCCGAACGGCATCCCGGGTGAAAAAGGTCCGGCGGGTGAAAGAGGTGCCCCTGGTCCGGGTCCCAGCGGTCCGCGTGGCCAACCTGGCGTCATGGGTTTTCCGGGCCCGAAAGGCCCGCGTGGTGAACGCGGAGAGGCAGGTATTCCGGGTGTGCCGGGTGCCAAGGGTGAAGATGGCAAGGACGGCAGCCCGGGCGAACCGGGTGCCAACGGTCTGCCGGGTGCGGCAGGCGAGCGTGGTGCGCCAGGATTCCGCGGCCCGGCTGGCCCGAACGGCATCCCGGGTGAAAAGGGCCCGGCGGGCGAGCGCGGTGCGCCAGGCCCGGGACCGTCTGGTCCGCGTGGCCAGCCGGGTGTGATGGGTTTCCCGGGCCCGAAAGGCCCGCGTGGAGAACGTGGCGAAGCAGGTATTCCGGGTGTTCCAGGTGCGAAAGGTGAGGACGGCAAGGATGGTAGCCCGGGCGAGCCGGGCGCGAATGGTTTGCCTGGCGCTGCTGGCGAGCGCGGTGCGCCGGGTTTTCGTGGTCCAGCTGGCCCGAATGGTATCCCGGGCGAGAAGGGCCCTGCCGGCGAGCGTGGTGCGCCAGGTCCGCTCGAGCACCACCACCACCACCACTGA
Claims (9)
1. a gene of human collagen, which is characterized in that the gene sequence has the sequence table SEQ ID: the nucleotide sequence shown in NO. 1.
2. A method for producing human collagen, comprising the steps of:
constructing escherichia coli genetic engineering bacteria;
fermenting and culturing escherichia coli genetic engineering bacteria;
inducing and expressing recombinant human collagen;
purification of recombinant human collagen.
3. The method for producing human-derived collagen according to claim 2, wherein:
the construction method of the escherichia coli genetic engineering bacteria comprises the following steps:
optimally selecting DNA fragments of human III type collagen genes, and synthesizing recombinant plasmids;
transferring the recombinant gene into an escherichia coli expression strain, and screening to obtain escherichia coli genetic engineering bacteria.
4. A method for producing human collagen according to claim 3, wherein:
the method for fermenting and culturing the escherichia coli genetic engineering bacteria comprises the following steps:
adding 0.1%50 mug/mL kana antibiotic into LB liquid culture medium, picking single colony, mixing uniformly, and culturing in a shaking table at 37 ℃ and 220rpm for 12h;
adding 0.1%50 μg/mL kana antibiotic into LB liquid culture medium, adding 1.0% of overnight culture bacterial liquid, placing in a shaking table at 37deg.C and 220rpm for culturing, adding inducer IPTG for induction after OD600 value is 0.4-0.6 when logarithmic growth phase is measured, and inducing for 24h at low temperature of 16deg.C and 220 rpm.
5. The method for producing human-derived collagen according to claim 4, wherein:
the method for purifying the recombinant human collagen comprises the following steps:
resuspending the bacteria with Tris buffer, sonicating, centrifuging and collecting supernatant;
and purifying the supernatant by using a nickel ion affinity column to obtain the recombinant human collagen.
6. The detection method of the human collagen is characterized by comprising the following steps of:
carrying out MTT experiments on the humanized collagen to obtain MTT experiment results;
performing a cell scratch experiment on the humanized collagen to obtain a cell scratch experiment result;
cell adhesion measurement and relative adhesion calculation were performed on human collagen to obtain calculation results.
7. The method for detecting human collagen according to claim 6, wherein:
the method for carrying out MTT experiment on the humanized collagen and obtaining MTT experiment results comprises the following steps:
BALB/3T3 cells were incubated at 37℃with 5% CO 2 The cells were cultured in a cell incubator, and the cell density and status were observed under an inverted microscope every day. Cell passage or cell seeding is performed when the cells grow to 80-90% of the flask. Cells were grown at 3X 10 using complete medium 3 Planting the seeds/holes in a 96-well plate, setting four compound holes at 100 mu L of each hole, culturing for 24 hours at 37 ℃ under the condition of 5% carbon dioxide, and then changing the culture solution into a maintenance culture solution for starvation treatment for 24 hours;
the maintenance culture solution was discarded, and 100. Mu.L of recombinant collagen prepared in the maintenance medium was added thereto at a final concentration of 0mg/mL, 0.01mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, respectively. The cells were cultured at 37℃under 5% carbon dioxide for 72 hours. The blank control group is only added with a maintenance medium;
10. Mu.L of MTT solution was added to each well, and the mixture was incubated at 37℃for 4 hours under 5% carbon dioxide. Carefully sucking and discarding the supernatant in the holes, adding 100 mu LDMSO into each hole, fully blowing and uniformly mixing, then placing into an enzyme labeling instrument, measuring the light absorption value at the wavelength of 570nm, and obtaining the MTT experiment result.
8. The method for detecting human collagen according to claim 6, wherein:
the method for carrying out cell scratch experiments on the humanized collagen and obtaining the cell scratch experiment result comprises the following steps:
marking three lines longitudinally for each well behind the 6-well plate with a marker pen. About 10X 10 per well 5 And (3) a plurality of cells. The goal is to reach a confluence state of 95-100% after 24 hours of culture;
after cell culture for 24h, the well plate was vertically aligned with a 10. Mu.L gun head to form scratches by pushing down the longitudinal streaks, the cells were rinsed 3 times with D-PBS, the scraped cells were removed, and 2mL of recombinant collagen prepared in maintenance medium was added to 6 wells at final concentrations of 0mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, respectively. The blank control group is only added with a maintenance medium;
at 37 ℃,5% CO 2 Culturing in an incubator, taking the intersection point of transverse and longitudinal scribing lines as a core after 0h and 24h, taking pictures under a 40-time microscope, and obtaining photos of 9 parts per hole;
measuring the area of the scratch area, and calculating the mobility of each group of cells by dividing the total area of the transitional cells of the fixed scratch area by the initial area of the fixed scratch area;
and obtaining the cell scratch experiment result.
9. The method for detecting human collagen according to claim 6, wherein:
the method for carrying out cell adhesion measurement and relative adhesion calculation on the human collagen comprises the following steps of:
100. Mu.L of recombinant collagen samples were added to each 96-well plate; incubate for 4h at 37℃in a 5% CO2 incubator. Excess D-PBS coating solution was removed from the wells, and 100. Mu.L of 0.5% BSA-PBS solution was added and incubated in a 5% CO2 incubator at 37℃for 1h. After removing the liquid in the wells, washing three times with D-PBS, discarding the cleaning solution, sealing with a sealing film, and standing at 4 ℃ for later use. Before use, placing the cell plate in an incubator for temperature return;
BALB/3T3 cells were cultured at 37℃in a 5% CO2 cell incubator, and the cell density and status were observed under an inverted microscope every day. Cell passage or cell seeding is performed when the cells grow to 80-90% of the flask. Cells were grown at 5X 10 using complete medium that had been pre-mixed with Hoechst33342 fluorescent stain 4 Each well was seeded in a 96-well plate with 100. Mu.L of each well, three duplicate wells were set, covered with tin foil and incubated at 37℃with 5% CO 2. Incubation time is 1h;
fluorescent photographing was performed on each sample well before centrifugation. After photographing, each well was filled with 250 μ L D-PBS to form a "reverse meniscus" and covered with a sealing membrane, placed upside down, centrifuged at 250 centrifugal force for 5min, the sealing membrane was discarded and the supernatant was removed from the well. After washing 1 time with D-PBS, 100. Mu. L D-PBS was added, and each well was photographed;
and calculating and obtaining a calculation result.
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