JP7459362B1 - Recombinant humanized collagen and its applications - Google Patents

Abstract

[Problem] The present invention provides a recombinant humanized collagen and its application. [Solution] The recombinant humanized collagen comprises a leader peptide and at least one basic repeating unit, and the basic repeating unit comprises the amino acid sequence shown in SEQ ID No. 1. The present invention further provides a method for preparing the recombinant humanized collagen. The recombinant humanized collagen described in the present invention has good solubility, stability, biological activity and adhesiveness, is less immunogenic and safer, has low preparation costs, high efficiency, mature technology, is easy to operate, and has important application prospects. [Selected Figure] Figure 2

Description

The present invention belongs to the technical field of recombinant proteins, and in particular relates to a type of recombinant humanized collagen and its applications, which contains a leader peptide and at least one basic repeating unit.

Collagen is the most abundant protein in the animal body, accounting for approximately 30% of all proteins in the body. Between the epidermis and dermis, there is a single layer of tissue called the "cushion network", which contains a large amount of collagen, supports the epidermis, prevents the skin from sagging, and tightens the skin. It can be smoothed out. Since replenishment of type III collagen is the key to skin care, collagen can be widely applied in industries such as medicine and cosmetics.

However, natural collagen is generally insoluble in water, difficult to be used by the human body, has complex components, and has low safety. Currently commercially available animal-derived collagen carries the risk of infectious diseases and is more likely to cause immune rejection when used by humans. More importantly, animal-derived collagen is usually obtained through acid, alkali, and enzymatic degradation processes, and its biological activity is much lower than that of the human body's natural collagen. Human-derived collagen can be extracted from human placenta, but the source is limited and there are many limitations.

The structure of recombinant humanized collagen is similar to that of human-derived collagen, and after design and optimization based on human-derived collagen, it can enhance collagen activity and reduce immunogenicity.

CN107714504A discloses a human-derived collagen composition comprising, in weight percent, 0.001-0.5% of the same human-derived collagen, 0.2-20.0% of a botanical conditioner, and a functional combination. The human-derived collagen structural amino acid sequence contains 599 amino acids and a molecular weight of 55.0 kDa. The composition has synergistic effects such as moisturizing, transdermal absorption, skin care, anti-inflammatory, hypoallergenic, nourishing and repairing, and antibacterial benefits. The human-derived collagen has wide application prospects in the field of skin care products, but the human-derived collagen has a large molecular weight and poor stability, making it difficult to maintain biological activity over a long period of time. This leads to the skin care effects not lasting long.

Therefore, it is an urgent issue to provide human type III collagen that is highly soluble, has low immunogenicity, has high biological activity, and is easily available, as well as a method for its preparation.

China Patent Application Publication No. 107714504

In response to the deficiencies of the prior art and practical needs, the present invention provides a recombinant humanized collagen and its application, which has good solubility, stability and biological activity, high bioadhesiveness, cell healing promotion ability and proliferation ability, low immunogenicity, and practical value.

To achieve this objective, the present invention adopts the following technical scheme.

In aspect 1, the present invention comprises:
comprising a leader peptide and at least one basic repeat unit;
The basic repeating unit is SEQ ID No. Containing the amino acid sequence shown in 1,
Recombinant humanized collagen is provided.

Here, the number of basic repeating units is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and within the numerical range. Any other specific point value can be selected and will not be described here.

SEQ ID No. 1: GPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPC.

In the present invention, the immunogenicity of recombinant collagen is reduced by adding a human-derived leader peptide, and the stability of recombinant collagen is increased by selecting a fragment with a crystallographic structure in type III collagen as the basic repeating unit. did. The recombinant humanized collagen can be expressed in vitro by protein expression technology, the production cost is low, the product has a single component, is safer, and has good solubility and adhesive properties. However, it has good biological activity and has wide application prospects in related fields such as medicine and cosmetics.

In the present invention, the recombinant humanized collagen has a single chain structure and exhibits soluble expression.

Preferably, the leader peptide has SEQ ID no. It contains the amino acid sequence shown in 2.

SEQ ID No. 2: MESCPTGPQNYSPQYDSYDVKSGVAVG.

Preferably, the number of said elementary repeating units is 3 to 6, and may be, for example, 3, 4, 5 or 6.

In the present invention, by controlling the number of basic repeating units within the range of 3 to 6, the recombinant protein can have both good stability and solubility, and when the number of basic repeating units is small, , the stability of the recombinant protein is poor, and the number of basic repeating units is too large, which reduces the solubility of the recombinant protein.

Preferably, the recombinant humanized collagen comprises the amino acid sequence shown in SEQ ID No. 3.

SEQ ID No. 3:
MESCPTGPQNYSPQYDSYDVKSGVAVGGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSE GSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCHHHHHHH.

In aspect 2, the invention provides a nucleic acid molecule encoding the recombinant humanized collagen according to aspect 1.

Preferably, the nucleic acid molecule has SEQ ID no. It contains the nucleotide sequence shown in 4.

SEQ ID No. 4:
atggaatcttgccgacggtccgcagaactactctccgcagtacgactcttacgacgttaaatctggtgttgctgttggtggtccgatcggtccgccgggtccgcgtggtaaccgtggtgaa cgtggttctgaaggttctccgggtcacccgggtcagccggggtccgccgggtccgccggtgctccgggtccgtgcggtccgatcggtccgccgggtccgcgtggtaaccgtggtgaacgtggttc tgaaggttctccgggtcacccgggtcagccgggtccgccgggtccgccgggtgctccgggtccgtgcggtccgatcggtccgccgggtccgcgtggtaaccgtggtgaacgtggttctgaaggtt ctccgggtcacccgggtcagccgggtccgccgggtccgccgggtgctccgggtccgtgcggtccgatcggtccgccggtccgcgtggtaaccgtggtgaacgtggttctgaaggttctccgggt cacccgggtcagccgggtccgccgggtccgccgggtgctccgggtccgtgccaccatcatcaccaccattaa.

In the present invention, the nucleic acid molecule sequence encoding the recombinant humanized collagen is codon-optimized based on the codon bias of the host, thereby significantly improving the expression efficiency in E. coli.

In the present invention, Nde I and Xho I enzyme cleavage sites are introduced into the nucleic acid molecule sequence and used for ligation to an expression vector.

In aspect 3, the invention provides an expression vector comprising at least one copied nucleic acid molecule according to aspect 2.

In aspect 4, the present invention provides a recombinant cell that expresses the recombinant humanized collagen described in aspect 1.

Preferably, the nucleic acid molecule according to aspect 2 is integrated into the genome of the recombinant cell.

Preferably, the recombinant cell contains the expression vector described in aspect 3.

In aspect 5, the present invention provides:
constructing an expression vector, introducing the expression vector into recipient cells, and selecting;
Cultivating the selected engineered bacterial strain, inducing expression and purifying it to obtain the recombinant humanized collagen;
A method for preparing recombinant humanized collagen according to aspect 1 is provided.

Preferably, the receptor cell comprises an E. coli expression strain.

Preferably, the method of inducing expression includes IPTG induction.

Preferably, the method further includes a step of disrupting the bacterial cells before the purification.

Preferably, the method of purification comprises nickel ion column affinity chromatography.

As a preferred technical proposal, the method for preparing recombinant humanized collagen according to the present invention includes:
(1) Linking the nucleic acid molecule encoding the recombinant humanized collagen to a plasmid vector to construct an expression vector, introducing the expression vector into an E. coli expression strain, and selecting to obtain genetically recombinant E. coli. and,
(2) fermenting and culturing the genetically modified E. coli, inducing protein expression with IPTG, and collecting the bacterial cells by centrifugation;
(3) Ultrasonic disruption of the collected bacterial cells and collection of the supernatant by centrifugation;
(4) performing nickel ion column affinity chromatography purification on the supernatant to obtain the recombinant humanized collagen;

In aspect 6, the invention provides the application of the recombinant humanized collagen according to aspect 1 in the preparation of cosmetics or skin damage treatment agents.

Compared with the prior art, the present invention has the following beneficial effects.

The present invention introduces a basic repeating unit into recombinant humanized collagen to improve the solubility of collagen, increase the stability of recombinant protein, and reduce the immunogenicity of the protein by adding a human-derived leader peptide. The recombinant humanized collagen can be synthesized in vitro by a protein expression system, with high production yield, low cost, mild reaction conditions, and no chemicals for recombinant protein activity. reduce the impact of Collagen is a major component of the extracellular matrix. The main biological function of recombinant humanized collagen is to provide a scaffold and a favorable microenvironment for cells, such as promoting cell adhesion, proliferation growth, migration, etc. Therefore, the biological function of recombinant collagen can be evaluated by evaluating cell-collagen interactions. The cell adhesion activity of the recombinant humanized collagen of the present invention is superior to that of natural human collagen, and it has good cell migration activity and the ability to promote cell proliferation and growth, as well as thermal stability. It has better biological activity, stronger adhesiveness, and good adhesiveness can improve the adhesion with the skin, and has important application prospects in the field of wound surface repair. The method for preparing recombinant humanized collagen is technologically mature and easy to manipulate, facilitating the widespread use and use of related products.

1 is a plasmid map of the expression vector in Example 1. 1 is a diagram showing the detection results of SDS-PAGE of recombinant humanized collagen in Example 1, in which lane M is a standard protein molecular weight marker and lane 1 is recombinant humanized collagen. It is a detection result diagram of relative cell adhesion activity in Test Example 1 of the present invention. It is a detection result of cytotoxicity in Test Example 2 of the present invention. It is a detection result of the cell scratch in Test Example 2 of this invention. 1 shows the results of cell proliferation detection in Test Example 3 of the present invention. This is the detection result of SDS-PAGE electrophoresis of recombinant humanized collagen in Test Example 4 of the present invention. In the figure, lane M is a standard protein molecular weight marker, and lane 1 is the recombinant humanized collagen before heating. and lane 2 is the recombinant humanized collagen after heating.

In order to further explain the technical means used in the invention and its effects, the invention will be further explained below with reference to examples and drawings. It will be understood that the specific embodiments described herein are merely for interpretation of the present disclosure and are not intended to limit the invention.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature within the field or according to product instructions are followed. Any reagents or equipment used that do not specify the manufacturer are normal products purchased through legitimate channels.

In this example, recombinant humanized collagen is prepared, the recombinant humanized collagen includes a leader peptide and four basic repeating units, and the sequence of the recombinant humanized collagen is SEQ ID No. As shown in 3.

SEQ ID No. 3:
MESCPTGPQNYSPQYDSYDVKSGVAVGGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSE GSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCHHHHHHH.

The recombinant humanized collagen was prepared by the following method.

(1) Codon optimization was performed on the nucleic acid molecule encoding the recombinant humanized collagen, Nde I and Xho I enzyme cleavage sites were introduced, and SEQ ID No. The nucleotide sequence shown in 4 was obtained.

SEQ ID No. 4:
atggaatcttgccgacggtccgcagaactactctccgcagtacgactcttacgacgttaaatctggtgttgctgttggtggtccgatcggtccgccgggtccgcgtggtaaccgtggtgaa cgtggttctgaaggttctccgggtcacccgggtcagccggggtccgccgggtccgccggtgctccgggtccgtgcggtccgatcggtccgccgggtccgcgtggtaaccgtggtgaacgtggttc tgaaggttctccgggtcacccgggtcagccgggtccgccgggtccgccgggtgctccggtccgtgcggtccgatcggtccgccgggtccgcgtggtaaccgtggtgaacgtggttctgaaggtt ctccgggtcacccgggtcagccgggtccgccgggtccgccgggtgctccgggtccgtgcggtccgatcggtccgccggtccgcgtggtaaccgtggtgaacgtggttctgaaggttctccgggt cacccgggtcagccgggtccgccgggtccgccgggtgctccgggtccgtgccaccatcatcatcaccaccattaa

(2) SEQ ID No. The nucleotide sequence shown in 4 and the plasmid vector pET-32a(+) were ligated after double enzyme digestion with Nde I and Xho I, and E. coli DH5α was transformed. Monoclonal colonies were picked up and then alkali The plasmid was extracted by the lysis method, positive clones were accurately identified by sequencing, and the expression vector was successfully constructed and the plasmid map is shown in FIG. 1.

(3) The expression vector was introduced into E. coli expression strain OverExpress C43 (DE3) and selected to obtain recombinant E. coli.

(4) Pick up the monoclonal colonies after selection, culture them overnight in 4 mL of LB medium, and culture them at 37°C in a shake flask with a 2% inoculation amount until the _OD600 reaches 0.4 to 0.6. Then, IPTG was added at a ratio of 1:5000, cultured at 30°C for 15 to 20 hours to induce protein expression, and the bacterial cells were collected by centrifugation.

(5) Resuspend the bacterial cells in a centrifuge tube using 50 mmol of Tri-HCl solution (pH 7.4), crush the bacterial cells by ultrasonication in an ice-water mixture environment (run for 2 s, stop for 4 s), and perform 6000 rpm. The mixture was centrifuged for 30 min and the supernatant was collected.

(6) The supernatant was purified using a nickel ion affinity column in a protein purification device to obtain the recombinant humanized collagen, and the SDS-PAGE detection results are as shown in FIG.

In this example, the recombinant humanized collagen was named COL-HJ-01.

[Comparative example 1]
This comparative example provided type I collagen derived from human placenta, which was purchased from Sigma and had the product number C7774.

[Comparative example 2]
This comparative example provided type III collagen derived from human placenta, was purchased from Sigma, and had the product number C4407.

[Test Example 1]
In this test example, the cell adhesion of the recombinant humanized collagen prepared in Example 1 and the natural human placenta-derived collagen of Comparative Examples 1 and 2 was tested, and the steps were as follows.

(1) Plating protein samples: Protein samples were added to a 96-well plate at 100 μL in each well, and no protein samples were added to blank wells.

(2) Coating of protein sample: The above 96-well plate was placed in a 37° C., 5% CO ₂ incubator and coated.

(3) Blocking of protein samples: Add 100 μL of 1% BSA-PBS solution, block in a 5% CO ₂ incubator at 37°C, remove excess blocking solution, and wash 3 times with D-PBS. .

(4) Preparation of cells: Prepare a Bal b/c3T3 cell suspension of 5×10 ⁴ cells/mL, add 100 μL to each well in a 96-well plate, culture for 1 to 4 h, and incubate each well 6 times. has been established.

(5) Detection: After culturing, detection was performed using the MTT method, and absorbance values were detected at wavelengths of 570 nm and 630 nm.

(6) Calculation: Calculate the relative cell adhesion activity of the protein sample, sample cell adhesion rate=OD value of sample group/OD value of blank group.

The results are shown in FIG. 3. In FIG. 3, the positive control is the human-derived collagen in Comparative Example 1 and Comparative Example 2, and COL-HJ-01 is the recombinant human collagen prepared in Example 1. The collagen was COL-HJ-01. As can be seen from the figure, the recombinant humanized collagen COL-HJ-01 prepared in Example 1 of the present application was superior to type I collagen and type III collagen extracted from human placenta. Possessing adhesive ability, it has exhibited good biological activity and can be applied in the preparation of related medicines and cosmetics.

[Test Example 2]
In this test example, cytotoxicity and cell scratch experiments were conducted on the recombinant humanized collagen prepared in Example 1.

(1) Preliminary experiment (cytotoxicity experiment)
Cytotoxicity was detected. Find a suitable solvent to dissolve the sample, generally the order of solvents of choice is ultrapure water, 95% ethanol, DMSO. The initial experiment was performed with the maximum solubility as the highest concentration, and the sample concentration range for the formal experiment was reduced according to the circumstances of the initial experiment, and the safe concentration used for the formal experiment was selected. As shown in FIG. 4, the results of the cytotoxicity experiment showed that COL-HJ-01 had no cytotoxicity.

(2) Formal experiment (cell scratch experiment)
(a) 2 mL of diluted cell suspension was added to a 6-well plate, and the concentration was set to 1×10 ⁶ cells/well.

(b) After the cells had attached to the walls and proliferated to approximately 80-90% of the 6-well plate, they were pretreated with DMEM medium containing mitomycin C for 2 hours.

(c) After making a wound with a 10 μL pipetman and washing three times with PBS, DMEM medium containing mitomycin C was added and the sample was added.

(d) After adding the sample, the scratch width was photographed and recorded under an inverted microscope at 0 h, and the sample was then placed in an incubator and cultured for 24 h.

(e) After 24 h, the 6-well plate was removed from the CO ₂ incubator and placed under an inverted microscope to photograph the scratch width for 24 h and record the data.

(f) Calculation: Scratch healing rate (%)=(S ₀ −S ₂₄ scratch area)/S ₀ scratch area×100%.

As shown in FIG. 5, in this experiment, the sample group within the detection concentration range had a significant difference compared to the Control group (blank), and had the effect of promoting cell migration.

[Test Example 3]
This test example is an experiment to test the cell proliferation of the recombinant humanized collagen prepared in Example 1, and the experimental steps are as follows:

(1) 100 μL of the diluted cell suspension was added to a 96-well plate, and the concentration was set to 1.0×10 ⁴ cells/well.

(2) The cells were placed in a CO ₂ incubator and cultured for 24 hours.

(3) After adding the sample, it was placed in an incubator and cultured for 24 hours.

(4) A 10-fold dilution of the CCK-8 reagent was prepared in the medium.

(5) After thoroughly discarding the sample-containing medium in the 96-well plate, 100 μL of the above diluted CCK-8 solution was added to each well, and cultured in a CO ₂ incubator for 1 to 2 hours.

(6) The 96-well plate was taken out and the absorbance was measured at 450 nm using a microplate reader.

HaCaT cell viability (%) = 100% x absorbance value of blank group/absorbance value of sample group.

As shown in FIG. 6, in this experiment, the sample group within the detection concentration range had a significant difference compared to the control group (blank), and had the effect of promoting cell proliferation.

[Test Example 4]
This test example is an experiment to test the thermal stability of the recombinant humanized collagen prepared in Example 1, and the experimental steps are as follows.

The present thermal stability experiment used a water bath thermal stimulation method, in which the samples were treated in a 60°C water bath for 15 minutes, centrifuged after heating, and the supernatant was subjected to SDS-PAGE. As shown in FIG. 7, lane M was the standard protein molecular weight marker, lane 1 was the recombinant humanized collagen before heating, and lane 2 was the recombinant humanized collagen after heating. Water bath heating precipitated a large amount of contaminant proteins, but there was still an obvious band in COL-HJ-01, indicating that the protein has thermostable performance. It has stable advantages in subsequent application and transportation.

In summary, the recombinant humanized collagen prepared in this application has both good solubility and thermal stability, low immunogenicity, good safety, high biological activity, and strong adhesiveness. , the ability to promote cell healing and proliferation is strong, the technology of the preparation method is mature, the efficiency is high, the cost is low, it can be produced in mild conditions, the use of chemical reagents is reduced, and it is applied in actual production. It's worth it.

Although the detailed method of the present invention has been explained through the above embodiments, the present invention is not limited to the above detailed method, that is, the present invention must be carried out depending on the above detailed method. The applicant declares that this does not mean that it will not be possible. Those skilled in the art will understand that any improvements to the present invention, equivalent substitutions and additions of auxiliary ingredients to the raw materials of the products of the present invention, selection of specific forms, etc., are all within the protection scope and disclosure scope of the present invention. You should understand that.

Claims (7)

A recombinant humanized collagen comprising:
The amino acid sequence of the recombinant humanized collagen is shown in SEQ ID No. 3.
A recombinant humanized collagen characterized by: 1. A nucleic acid molecule comprising:
Encoding the recombinant humanized collagen of claim 1,
The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 4.
A nucleic acid molecule comprising: comprising at least one copied nucleic acid molecule according to claim 2;
An expression vector characterized by: Expressing the recombinant humanized collagen according to claim 1.
A recombinant cell characterized by: The nucleic acid molecule of claim 2 is integrated into the genome of the recombinant cell.
The recombinant cell according to claim 4. The recombinant cell contains the expression vector according to claim 3.
The recombinant cell according to claim 4, characterized in that: 2. Use of the recombinant humanized collagen according to claim 1 in the preparation of a cosmetic or a skin damage treatment drug.