An extracellular matrix collagen for scalp care, its preparation method and application

Abstract

The present application discloses an extracellular matrix collagen for scalp care, its preparation method and application, relating to the technical field of genetic engineering. An extracellular matrix collagen for scalp care, wherein the amino acid sequence of the extracellular matrix collagen is as shown in Seq ID NO.1. In the present application, a variety of different subtypes of collagen are fused by synthetic biology methods according to the functions of collagen, and their amino acid sequences and gene sequences are optimized to enable their highly efficient expression in Saccharomyces cerevisiae, thereby obtaining the extracellular matrix collagen for scalp care in the present application. It retains biological activities superior to natural collagen and has more significant effects in promoting hair growth and scalp repair. It can be applied to hair growth and anti - hair loss products and has the great advantage of having no side effects.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and particularly relates to an extracellular matrix collagen for scalp care, its preparation method and application. Background Art

[0002] The scalp extracellular matrix (ECM) refers to a complex network present in scalp tissues, including various components such as multiple proteins, glycosaminoglycans, and glycoproteins, which play important roles in maintaining scalp structure, supporting cell proliferation and migration, and participating in physiological processes such as healing and regeneration. Generally, the scalp mainly contains several types of collagen: Type I collagen, which is the most abundant type of collagen in the scalp, mainly responsible for providing structural strength and support, and is widely present in skin, bones, tendons, and ligaments; Type III collagen, which coexists with Type I collagen and mainly plays a role in the elasticity and toughness of the skin. The proportion of Type III collagen is relatively high in young skin, and its content usually decreases with age; Type IV collagen, which is mainly present in the basement membrane and plays a role in supporting cells and tissues; Type VII collagen, which exists between the dermis and the epidermis and participates in the stability of skin structure. These proteins and other components together constitute the extracellular matrix of the scalp and maintain the health and function of the scalp.

[0003] There are a variety of existing hair growth products on the market. Some of them can show effects in the short term, but the effects weaken after long-term use and cannot be maintained persistently. Moreover, some of them may contain irritating ingredients or chemicals, which may cause side effects such as skin irritation, allergic reactions, and increased hair loss after use, especially for people with sensitive skin. Using collagen for scalp care can effectively solve problems such as short-term effects and allergies. However, there are a variety of collagens in the skin extracellular matrix, and different types of collagen often have different effects. The effect of a single type of collagen is limited, and it may need to be used in combination with a specific frequency or other products, thus limiting its application in hair growth and anti-hair loss products. Summary of the Invention

[0004] The main purpose of this application is to provide an extracellular matrix collagen for scalp care, its preparation method and application, aiming to solve the technical problem that the existing collagen products have limited effects on promoting hair growth.

[0005] To achieve the above object, this application proposes an extracellular matrix collagen for scalp care, and the amino acid sequence of the extracellular matrix collagen is as shown in Seq ID NO.1.

[0006] Optionally, the gene sequence of the extracellular matrix collagen is as shown in Seq ID NO.2.

[0007] This application also provides a method for preparing extracellular matrix collagen for scalp care, comprising the following steps:

[0008] Construct a recombinant plasmid pYES2 / CT-MFα-ECMCOL according to the gene sequence shown in Seq ID NO.2 and the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα;

[0009] Electrotransform the recombinant plasmid pYES2 / CT-MFα-ECMCOL into Saccharomyces cerevisiae INVSc1 competent cells, culture them, and then perform PCR amplification and identify positive clone strains to obtain the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL;

[0010] Induce the expression of the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL and collect the supernatant of the induced expression;

[0011] After purifying the supernatant of the induced expression, obtain the extracellular matrix collagen stock solution.

[0012] Optionally, the step of constructing the recombinant plasmid pYES2 / CT-MFα-ECMCOL according to the gene sequence shown in Seq ID NO.2 and the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα includes:

[0013] Synthesize a gene fragment according to the gene sequence shown in Seq ID NO.2, and insert the synthesized gene fragment into the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα through the restriction enzyme sites Not Ⅰ and Xba I to obtain the recombinant plasmid pYES2 / CT-MFα-ECMCOL.

[0014] Optionally, the step of electrotransforming the recombinant plasmid pYES2 / CT-MFα-ECMCOL into Saccharomyces cerevisiae INVSc1 competent cells and culturing them includes:

[0015] Add the recombinant plasmid pYES2 / CT-MFα-ECMCOL to Saccharomyces cerevisiae INVScl competent cells, mix well, let it stand on ice, perform electrotransformation, then add a pre-cooled sorbitol solution, mix well, incubate at 30°C for 1 h, then centrifuge and discard the supernatant, resuspend and spread on an SC-U solid plate, and culture it inverted at 30°C until single clone colonies grow.

[0016] Optionally, the step of performing PCR amplification and identifying positive clone strains to obtain the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL includes:

[0017] Pick the monoclonal colonies grown on the SC-U solid plate, inoculate them into the YPD liquid medium, and culture them overnight at 30 °C and 180 rpm. Then, select 10 monoclonal colonies after overnight culture, extract genomic DNA respectively, amplify the target gene by PCR, and identify the positive clone strains by SDS-PAGE electrophoresis to obtain the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL.

[0018] Optionally, in the step of PCR amplification, the PCR conditions are: pre-denaturation at 98 °C for 5 min, thermal denaturation at 98 °C for 50 s, annealing at 60 °C for 30 s, extension at 72 °C for 60 s, for 35 cycles; final extension at 72 °C for 10 min.

[0019] Optionally, the step of inducing the expression of the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL and collecting the supernatant of the induced expression includes:

[0020] Pick the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL and inoculate it into the SC-U selective medium, culture it with shaking at 30 °C and 220 rpm, measure the OD 600nm absorbance value, then transfer it to the SC-U induction medium to make the initial OD 600nm absorbance value 0.3 - 0.5, and continue the culture;

[0021] Add galactose to the SC-U induction medium every 24 h to a final concentration of 2.0%, and after centrifugation, collect the supernatant of the induced expression.

[0022] Optionally, the step of purifying the supernatant of the induced expression to obtain the extracellular matrix collagen stock solution includes:

[0023] Use a cation exchange medium to equilibrate the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remain unchanged. Load the supernatant of the induced expression, set the loading flow rate at 5 ml / min, start collecting samples when the A280 absorbance value detected by ultraviolet rises; after the loading is completed, re-equilibrate the chromatography column with phosphate buffer until the conductivity and ultraviolet A280 absorbance values are the lowest and no longer change, and stop collecting samples; then elute with phosphate buffer containing NaCl and collect the corresponding protein, and after dialysis, obtain the extracellular matrix collagen stock solution.

[0024] This application also proposes an application of the extracellular matrix collagen for scalp care, which is used in hair growth and anti-hair loss products after mixing the above extracellular matrix collagen with hyaluronic acid.

[0025] The present application has at least the following beneficial effects:

[0026] By comparing the conserved regions of 29 types of collagen, various different subtypes of collagen were fused using synthetic biology methods according to the functions of collagen, and their amino acid sequences and gene sequences were optimized to enable their efficient expression in Saccharomyces cerevisiae. The Saccharomyces cerevisiae expression system has no endotoxin, high yield, simple culture medium, stable expressed protein, and can secrete it extracellularly for easy purification. At the same time, it has functions such as correctly processing, modifying, and properly spatially folding heterologous proteins, and heterologously expressing the target protein. Thus, the extracellular matrix collagen for scalp care of the present application was obtained. Its amino acid sequence is shown as Seq ID NO.1, and its gene sequence is shown as Seq ID NO.2. It retains better activity than natural collagen, has low production cost and stable product quality. And through verification, compared with single-subtype collagen, the extracellular matrix collagen after fusion in the present application is expressed more efficiently, has more excellent cell adhesion promotion activity and cell proliferation promotion activity, higher biological activity, and more significant effects in promoting hair growth and scalp repair. It can be applied to hair growth and anti-hair loss products. And because this extracellular matrix collagen belongs to a biological protein and has the advantages of being easily decomposed and having no residue, it has a huge advantage of no side effects compared with ordinary chemical skin care products and hormonal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0028] Figure 1 It is the spectrum diagram of the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα described in the embodiment of the present application;

[0029] Figure 2 It is the SDS-PAGE electrophoresis result diagram of the positive clone strain described in the embodiment of the present application;

[0030] Figure 3 It is the SDS-PAGE electrophoresis result diagram of the induced expression supernatant described in the embodiment of the present application;

[0031] Figure 4 It is the SDS-PAGE electrophoresis result diagram of the purified protein described in the embodiment of the present application;

[0032] Figure 5It is a comparison chart of cell observation results under a 100-fold microscope in the detection of cell adhesion promoting activity according to the embodiments of the present application; among them, figure (a) is the cell observation result chart of the negative control well under a 100-fold microscope; figure (b) is the cell observation result chart of the sample well of the extracellular matrix collagen composition under a 100-fold microscope;

[0033] Figure 6 It is a result chart of the detection of cell adhesion promoting activity according to the embodiments of the present application;

[0034] Figure 7 It is a comparison chart of cell observation results under a 100-fold microscope in the detection of cell proliferation promoting activity according to the embodiments of the present application; among them, figure (c) is the cell observation result chart of the negative control well under a 100-fold microscope; figure (d) is the cell observation result chart of the sample well of the extracellular matrix collagen composition under a 100-fold microscope;

[0035] Figure 8 It is a result chart of the detection of cell proliferation promoting activity according to the embodiments of the present application;

[0036] Figure 9 It is a measurement result chart of hair coverage rate according to the embodiments of the present application.

[0037] The realization, functional features and advantages of the object of the present application will be further described with reference to the embodiments and the accompanying drawings. Detailed Embodiments

[0038] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0039] Sequence Listing Description (the sequence listing content is provided separately):

[0040] The amino acid sequence of the extracellular matrix collagen in the embodiments of the present application is shown in Seq ID NO.1;

[0041] The nucleotide sequence of the extracellular matrix collagen in the embodiments of the present application is shown in Seq ID NO.2.

[0042] Example 1 Preparation of Extracellular Matrix Collagen

[0043] 1 Sequence Selection

[0044] Collagen is divided into 29 subtypes. In this application, by comparing the conserved regions of 29 types of collagen, various different subtypes of collagen are fused by synthetic biology methods according to the functions of collagen. The Saccharomyces cerevisiae expression system is used, which has no endotoxin, high yield, simple culture medium, stable expressed protein and can be secreted extracellularly and is easy to purify. At the same time, it has functions such as correctly processing, modifying and properly folding heterologous proteins, and heterologously expressing the target protein, so as to obtain the extracellular matrix collagen for scalp care in this application. Its amino acid sequence is shown in Seq ID NO.1, which is derived from human collagen, and the sequence references are: UniProt 1277, UniProt QIP67963.1, UniProt AAH28178.1 and UniProtAAA58965.1.

[0045] Furthermore, according to the properties of the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα and the codon preference of the Saccharomyces cerevisiae host, the gene sequence encoding extracellular matrix collagen is optimized and designed, including reducing the repetition frequency of repetitive sequences and artificial design, etc., which helps to reduce errors in the translation process and increase the proportion of correctly folded proteins, thus obtaining the optimized gene sequence of extracellular matrix collagen, as shown in Seq ID NO.2.

[0046] 2 Construction and screening of recombinant yeast engineering bacteria

[0047] 2.1 Construction of recombinant plasmid

[0048] According to the gene sequence of extracellular matrix collagen shown in Seq ID NO.2, General Biology (Anhui) Co., Ltd. was commissioned to synthesize the gene fragment, and the synthesized gene fragment was inserted into the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα (as Figure 1 shown) through the restriction enzyme sites of Not Ⅰ and Xba I to obtain the recombinant plasmid pYES2 / CT-MFα-ECMCOL.

[0049] 2.2 Electroporation into Saccharomyces cerevisiae INVSc1 competent cells

[0050] Take 10 μL of the recombinant plasmid pYES2 / CT-MFα-ECMCOL and add it to 80 μL of Saccharomyces cerevisiae INVScl competent cells. After mixing, transfer it to a pre-chilled electroporation cuvette and let it stand on ice for 5 min. Adjust the Bio-Rad electroporator to the fungal setting, place the electroporation cuvette on the Bio-Rad electroporator for electroporation; after electroporation, quickly add 500 μL of pre-chilled 1 M sorbitol solution to the electroporation cuvette, mix well, incubate at 30 °C for 1 h, then centrifuge and discard 400 μL - 500 μL of the supernatant, resuspend the cells by pipetting and spread them on the SC-U solid plate; incubate the SC-U solid plate inverted at 30 °C until monoclonal colonies grow.

[0051] 2.3 Screening of positive clone strains

[0052] Pick monoclonal colonies growing on the SC-U solid plate and inoculate them into a centrifuge tube containing 500 μL of YPD liquid medium. Incubate overnight at 30 °C and 180 rpm. Then select 10 monoclonal colonies after overnight culture and extract genomic DNA respectively. After PCR amplification of the target gene, identify positive clone strains by SDS-PAGE electrophoresis. The electrophoresis identification results are as Figure 2 shown, (wherein, lane M: 100 bp Plus DNA Marker; lane 1: negative control; lanes 2 - 5: PCR products of monoclonal colonies), which is basically consistent with the theoretical extracellular matrix collagen band of 1130 bp.

[0053] Furthermore, select the positive clone strains with correct identification for the next experiment, and thus obtain the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL.

[0054] Specifically, the PCR conditions are: pre-denaturation at 98 °C for 5 min, thermal denaturation at 98 °C for 50 s, annealing at 60 °C for 30 s, extension at 72 °C for 60 s, 35 cycles; final extension at 72 °C for 10 min.

[0055] 3 Induced expression of the recombinant yeast engineering strain

[0056] Pick the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL and inoculate it into 20 mL of SC-U selective medium. Incubate with shaking overnight at 30 °C and 220 rpm, measure the OD 600nm absorbance value, calculate the corresponding volume of the bacterial liquid and transfer it to 100 mL of SC-U induction medium to make the initial OD 600nm absorbance value 0.3 - 0.5, and continue the culture;

[0057] Galactose was added to the SC-U induction medium every 24 h to a final concentration of 2.0%. After induction, the bacterial liquid was centrifuged to collect the supernatant of induced expression, and the expression level of the target protein was analyzed by SDS-PAGE electrophoresis. The results are as Figure 3 shown (where lane M: protein Marker; lane 1: supernatant before induction; lanes 2-4: supernatants of induced expression of different colonies). An obvious specific band could be observed at about 42 kDa. The recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL could produce extracellular matrix collagen of about 42 kDa after induction.

[0058] 4 Purification

[0059] A cation exchange medium (the chromatography packing material was SP Purose 6 High Performance produced by Qianchun, loaded on the GE Akta chromatography system) was used. The chromatography column was equilibrated with phosphate buffer (25 mM NaH2PO4, pH 4.0) until the conductivity value and the A280 absorbance value remained unchanged. The supernatant of induced expression was loaded, and the loading flow rate was set at 5 mL / min. The ultraviolet A280 absorbance value was detected. When it increased, sampling began; after the loading was completed, the cation chromatography medium was equilibrated with phosphate buffer until the conductivity and the ultraviolet A280 absorbance value were the lowest and no longer changed, and sampling was stopped; then it was eluted with NaH2PO4 buffer containing NaCl (1 M) and the corresponding protein was collected. The extracellular matrix collagen stock solution was obtained after dialysis. The expression level of the purified protein was analyzed by SDS-PAGE electrophoresis. The results are as Figure 4 shown. An obvious specific band could be observed at about 42 kDa, which was basically consistent with the predicted molecular weight.

[0060] Example 2 Preparation of the finished product of extracellular matrix collagen composition

[0061] The extracellular matrix collagen stock solution in Example 1 was mixed with hyaluronic acid to obtain the finished product of extracellular matrix collagen composition. Among them, the final concentration of extracellular matrix collagen was 4 mg / mL, and the final concentration of hyaluronic acid was 15%. This finished product of extracellular matrix collagen composition can be used in hair growth and anti-hair loss products.

[0062] Example 3 Detection of cell adhesion promoting activity

[0063] The extracellular matrix collagen composition in Example 2 was pre-diluted with PBS buffer to a total protein content of 0.5 μg / mL. After pre-dilution, it was serially diluted 2-fold in a 96-well plate, with a total of 8 dilution factors. 50 μL of the finished product samples of the extracellular matrix collagen composition at different dilution factors were added to each well. A negative control (without adding any protein) was set up, and 50 μL of PBS buffer was added as a control. At the same time, three single collagen control groups (all three single collagens were diluted to 0.5 μg / mL) were set up. After the same 2-fold dilution, different concentrations of the single collagen solutions were added to each well. Incubation was carried out overnight at 4 °C. After incubation, the liquid in the plate was discarded, and each well was washed with 100 μL of PBS, for a total of 3 washes. After washing, 100 μL of bovine serum albumin at 30 μg / μL was added to each well for blocking, and the plate was incubated in an incubator at 37 °C for 1 h. After incubation, the liquid in the plate was discarded, and each well was washed 3 times with 100 μL of PBS. Then, fibroblast cell suspension was added, and the cell seeding density was 1.0×10 5 cells / mL. 100 μL was seeded into each well, and the plate was incubated in an incubator for 5 h. The cell plate after incubation was washed 3 times with PBS, and the cell adhesion was observed under a 100-fold microscope. The results are as Figure 5 shown. Five points were selected under a 200-fold microscope, excluding the edge points, to count the number of adherent cells. According to the counting results, a curve was fitted to obtain the specific activity. The results are as Figure 6 shown.

[0064] As Figure 5 can be seen, only a very small number of cells survived in the negative control wells, indicating that the negative control experiment was valid. As Figure 6 can be seen, the extracellular matrix collagen composition of the present application has the activity of promoting fibroblast adhesion with type I collagen, type III collagen, and type IV collagen. However, compared with the three single collagens, the extracellular matrix collagen composition prepared in the present application has more excellent adhesion activity.

[0065] Example 4 Detection of promoting keratinocyte proliferation

[0066] The extracellular matrix collagen composition in Example 2 was pre-diluted with PBS buffer to a total protein content of 0.5 μg / mL. Human keratinocyte cell lines were cultured in DMEM medium with 10% fetal bovine serum at 37 °C and 5% CO2, and the cell concentration was controlled at 1.0×10 4 cells / mL - 5.0×10 4cells / mL and seeded into 96-well plates. During the seeding process, keep shaking constantly to ensure the same number of cells seeded in each well. Seed 100 μL in each well and culture at 37°C under 5% CO2 conditions; After 24 h, change to DMEM culture medium with 0.4% calf serum; After culturing for 24 h, discard the culture medium, add the pre-diluted finished product of the extracellular matrix collagen composition. At the same time, set up a negative control group (without adding any protein) and a single collagen control group (all three collagens are diluted to 0.5 μg / mL), with 2 replicate wells in each group, 100 μL in each well; Incubate at 37°C under 5% CO2 conditions for 68 h - 72 h; Add 20 μL of MTT solution to each well and culture at 37°C under 5% CO2 conditions for 5 h. The above operations are carried out under sterile conditions. After discarding the liquid in the culture plate, add 100 μL of dimethyl sulfoxide to each well. After fully dissolving and mixing evenly, observe the cell adhesion situation under a 100-fold microscope. The results are as Figure 7 shown. At the same time, on the microplate reader, with 630 nm as the reference wavelength and 570 nm as the test wavelength, measure the absorbance, record the measurement results, and process the experimental data using a computer program or the four-parameter regression calculation method. The results are as Figure 8 shown.

[0067] It can be seen from Figure 7 that only a very small number of cells survived in the negative control wells, indicating that the negative control experiment was successful; It can be seen from Figure 8 that the extracellular matrix collagen composition of the present application has the activity of promoting keratinocyte proliferation with type I collagen, type III collagen, and type IV collagen. However, compared with the three single collagens, the proliferation effect of the extracellular matrix collagen composition prepared in the present application is more excellent.

[0068] Example 5 Hair Growth Promotion Experiment

[0069] After removing the back hair of C57BL / 6 mice, they were divided into an experimental group and a control group. In the experimental group, the extracellular matrix collagen composition in Example 2 was applied externally to the wound surface of the mice. The control group was divided into 4 groups, and PBS, type I collagen, type III collagen, and type IV collagen were applied externally respectively. Use a digital camera to record the back hair of each mouse on the 0th day, 4th day, 8th day, and 12th day after treatment, and use Image J software to measure the hair area to calculate the hair coverage rate. The test results are as Figure 9 shown.

[0070] It can be seen from Figure 9 that the extracellular matrix collagen composition of the present application has the effect of promoting hair growth with type I collagen, type III collagen, and type IV collagen. However, the ability of the extracellular matrix collagen composition of the present application to promote hair growth is more excellent than that of several single collagens, and a higher hair coverage rate can be achieved in a short time.

[0071] In summary, in this application, by comparing the conserved regions of 29 types of collagens, multiple different subtypes of collagens were fused using synthetic biology methods according to the functions of collagens, and their amino acid sequences and gene sequences were optimized to enable their efficient expression in Saccharomyces cerevisiae. The Saccharomyces cerevisiae expression system has the advantages of being endotoxin-free, having a high yield, a simple culture medium, stable expression of proteins, and being able to secrete proteins extracellularly for easy purification. At the same time, it has the functions of correctly processing, modifying, and properly folding heterologous proteins in space. By expressing the target heterologous protein, the extracellular matrix collagen for scalp care in this application was obtained. Its amino acid sequence is shown in Seq ID NO.1, and its gene sequence is shown in Seq ID NO.2. It retains better activity than natural collagen, has a low production cost, and stable product quality. After verification, compared with single-subtype collagens, the extracellular matrix collagen fused in this application is expressed more efficiently, has more excellent cell adhesion promotion activity and cell proliferation promotion activity, higher biological activity, and more significant effects on promoting hair growth and scalp repair. It can be applied to hair growth and anti-hair loss products. Moreover, since this extracellular matrix collagen belongs to a biological protein and has the advantages of being easily decomposed and leaving no residue, it has a huge advantage of having no side effects compared with ordinary chemical skin care products and hormonal drugs.

[0072] The above are only optional embodiments of this application, and do not limit the patent scope of this application accordingly. Any equivalent structural transformation made using the content of the specification and drawings of this application under the inventive concept of this application, or any direct / indirect application in other related technical fields, is included in the patent protection scope of this application.

Claims

1. An extracellular matrix collagen for scalp care, characterized in that: The amino acid sequence of the extracellular matrix collagen is shown in Seq ID NO.1, and the gene sequence of the extracellular matrix collagen is shown in Seq ID NO.

2.

2. A method for preparing extracellular matrix collagen for scalp care according to claim 1, characterized in that: The following steps are involved: According to the gene sequence shown in Seq ID NO.2 and the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα, the recombinant plasmid pYES2 / CT-MFα-ECMCOL was constructed; The recombinant plasmid pYES2 / CT-MFα-ECMCOL is electrotransformed into Saccharomyces cerevisiae INVSc1 competent cells, cultured, and then amplified and identified positive clones by PCR to obtain the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL; Inducing the expression of the recombinant yeast engineering bacteria INVSc1 / pYES2 / CT-MFα-ECMCOL, and collecting the induced expression supernatant; After purifying the induced expression supernatant, an extracellular matrix collagen stock solution was obtained.

3. The method for preparing extracellular matrix collagen for scalp care according to claim 2, characterized in that: The step of constructing the recombinant plasmid pYES2 / CT-MFα-ECMCOL according to the gene sequence shown in Seq ID NO.2 and the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα comprises: The gene fragments were synthesized according to the gene sequence shown in Seq ID NO.2, and the synthesized gene fragments were inserted into the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα through the restriction sites Not Ⅰ and Xba I to obtain the recombinant plasmid pYES2 / CT-MFα-ECMCOL.

4. The method for preparing extracellular matrix collagen for scalp care according to claim 2, characterized in that: The step of electrotransforming the recombinant plasmid pYES2 / CT-MFα-ECMCOL into Saccharomyces cerevisiae INVSc1 competent cells and culturing the cells comprises: The recombinant plasmid pYES2 / CT-MFα-ECMCOL was added to the Saccharomyces cerevisiae INVScl competent cells, mixed, and allowed to stand on ice for electroporation transformation. The pre-cooled sorbitol solution was then added, mixed, and incubated at 30°C for 1 hour. The supernatant was discarded after centrifugation, blown evenly, and spread on a SC-U solid plate, and inverted and cultured at a constant temperature of 30°C until a monoclonal colony grew.

5. The method for preparing extracellular matrix collagen for scalp care according to claim 4, characterized in that: The step of obtaining the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL by PCR amplification and identification of positive clones includes: The monoclonal colonies grown on the SC-U solid plate were picked, inoculated into YPD liquid culture medium, and cultured overnight at 30°C and 180rpm. Ten monoclonal colonies after overnight culture were selected, and genomic DNA was extracted from each of them. After PCR amplification of the target gene, positive clones were identified by SDS-PAGE electrophoresis to obtain the recombinant yeast engineered bacteria INVSc1 / pYES2 / CT-MFα-ECMCOL.

6. The method for preparing extracellular matrix collagen for scalp care according to claim 5, characterized in that: In the PCR amplification step, the PCR conditions are: 98°C pre-denaturation for 5 min, 98°C thermal denaturation for 50 s, 60°C annealing for 30 s, 72°C extension for 60 s, 35 cycles; 72°C annealing for 10 min.

7. The method for preparing extracellular matrix collagen for scalp care according to claim 2, characterized in that: The step of inducing the expression of the recombinant yeast engineering bacteria INVSc1 / pYES2 / CT-MFα-ECMCOL and collecting the induced expression supernatant comprises: The recombinant yeast INVSc1 / pYES2 / CT-MFα-ECMCOL was selected and inoculated into SC-U selection medium, and cultured under shaking conditions of 30°C and 220 rpm. The OD 600nm absorbance value, and then transferred to SC-U induction medium to make the initial OD 600nm When the absorbance value is 0.3-0.5, continue culturing; Galactose was added to the SC-U induction medium every 24 hours to a final concentration of 2.0%, and the induced expression supernatant was collected after centrifugation.

8. The method for preparing extracellular matrix collagen for scalp care according to claim 2, characterized in that: The step of purifying the induced expression supernatant to obtain an extracellular matrix collagen stock solution comprises: A cation exchange medium was used, and the chromatography column was equilibrated with a phosphate buffer until the conductivity value and the A280 absorbance value remained unchanged. The induced expression supernatant was loaded, and the loading flow rate was set to 5 ml / min. When the ultraviolet A280 absorbance value increased, the sample was started; after the loading was completed, the chromatography column was re-equilibrated with a phosphate buffer until the conductivity and ultraviolet A280 absorbance values ​​were the lowest and no longer changed, and the sample was stopped; then the corresponding protein was eluted with a phosphate buffer containing NaCl and collected, and after dialysis, the extracellular matrix collagen stock solution was obtained.

9. A use of the extracellular matrix collagen for scalp care as claimed in claim 1, characterized in that: The extracellular matrix collagen is mixed with hyaluronic acid and used in the preparation of a hair growth and anti-hair loss product.

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