Extracellular matrix collagen for scalp care as well as preparation method and application of extracellular matrix collagen

Abstract

The invention discloses extracellular matrix collagen for scalp care as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. The invention relates to extracellular matrix collagen for scalp care. The amino acid sequence of the extracellular matrix collagen is shown as Seq ID NO. 1. According to functions of collagen, various different subtypes of collagen are fused by a synthetic biological method, and amino acid sequences and gene sequences of the collagen are optimized, so that the collagen is efficiently expressed in saccharomyces cerevisiae, and the extracellular matrix collagen for scalp care is obtained. The natural collagen retains the biological activity superior to that of natural collagen, has more remarkable effects on promoting hair growth and repairing scalp, can be applied to hair growth and hair loss prevention products, and has the huge advantage of no side effect.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to an extracellular matrix collagen protein for scalp care and a preparation method and application thereof. Background Art

[0002] The extracellular matrix (ECM) of the scalp refers to a complex network existing in the scalp tissue, including a variety of proteins, glycosaminoglycans and glycoproteins. It plays an important role in maintaining the scalp structure, supporting cell proliferation and migration, and participating in physiological processes such as healing and regeneration. Normally, the scalp mainly contains several types of collagen: type I collagen, which is the most abundant type of collagen in the scalp and is mainly responsible for providing structural strength and support. It is widely present in the skin, bones, tendons and ligaments; type III collagen, which exists together with type I collagen and mainly plays a role in the elasticity and toughness of the skin. The proportion of type III collagen is higher 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 the 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 many types of hair growth products on the market, but some of them can show effects in the short term, but the effects weaken after long-term use and cannot be maintained for a long time. They may also contain irritating ingredients or chemicals, which may cause skin irritation, allergic reactions, aggravated hair loss and other side effects after use, especially for people with sensitive skin. Using collagen to care for the scalp can effectively solve problems such as short-term effects and allergies, but there are rich types of collagen in the extracellular matrix of the skin, and different types of collagen often have different effects. The efficacy of a single type of collagen is limited and may require a specific frequency of use or other products, which limits 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 and a preparation method and application thereof, aiming to solve the technical problem that existing collagen products have limited efficacy in promoting hair growth.

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

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

[0007] The present application also proposes a method for preparing extracellular matrix collagen for scalp care, comprising the following steps: 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.

[0008] 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 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.

[0009] Optionally, the step of electrotransforming the recombinant plasmid pYES2 / CT-MFα-ECMCOL into Saccharomyces cerevisiae INVSc1 competent cells and culturing them 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.

[0010] Optionally, the step of amplifying and identifying positive clones by PCR to obtain the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL comprises: 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.

[0011] Optionally, 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.

[0012] Optionally, the step of inducing 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.

[0013] Optionally, 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.

[0014] The present application also proposes an application of extracellular matrix collagen for scalp care, wherein the extracellular matrix collagen is mixed with hyaluronic acid and used in a hair growth and anti-hair loss product.

[0015] This application includes at least the following beneficial effects: The present application compares the conserved regions of 29 collagens, fuses multiple different subtypes of collagen by synthetic biology methods according to the functions of collagen, and optimizes their amino acid sequences and gene sequences to enable efficient expression in Saccharomyces cerevisiae. The Saccharomyces cerevisiae expression system is endotoxin-free, has a high yield, a simple culture medium, stable expressed proteins that can be secreted to the extracellular space for easy purification, and has the functions of correctly processing, modifying, and reasonably spatially folding heterologous proteins to heterologously express the target protein, thereby obtaining the extracellular matrix collagen protein for scalp care of the present application, the amino acid sequence of which is shown in Seq ID NO.1, and the gene sequence is shown in Seq ID NO.2. It retains activity that is superior to natural collagen, has low production cost, and stable product quality. It has been verified that compared with a single subtype of collagen, the extracellular matrix collagen after fusion in the present application is more efficiently expressed, has better cell adhesion and cell proliferation activities, higher biological activity, and is more effective in promoting hair growth and scalp repair. It can be used in hair growth and anti-hair loss products. Moreover, since the extracellular matrix collagen is a biological protein, it has the advantage of being easy to decompose and leaving no residue. Compared with ordinary chemical skin care products and hormone drugs, it has the huge advantage of having no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0017] Figure 1 This is a spectrum of the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα described in the examples of this application; Figure 2 This is a diagram of the SDS-PAGE electrophoresis results of the positive clones described in the examples of this application; Figure 3 This is a diagram showing the SDS-PAGE electrophoresis result of the induced expression supernatant described in the examples of this application; Figure 4 This is a diagram of the SDS-PAGE electrophoresis result of the purified protein described in the examples of this application; Figure 5 The figures are comparison diagrams of cell observation results under a 100x microscope in the cell adhesion promotion activity detection described in the examples of the present application; wherein (a) is a diagram of cell observation results of a negative control well under a 100x microscope; (b) is a diagram of cell observation results of a sample well of an extracellular matrix collagen composition under a 100x microscope; Figure 6This is a graph showing the results of the cell adhesion promoting activity test described in the examples of the present application; Figure 7 The figures are comparison diagrams of cell observation results under a 100x microscope in the cell proliferation activity detection described in the examples of the present application; wherein, (c) is a diagram of cell observation results of a negative control well under a 100x microscope; (d) is a diagram of cell observation results of a sample well of an extracellular matrix collagen composition under a 100x microscope; Figure 8 This is a graph showing the results of the cell proliferation promoting activity test described in the examples of this application; Fig. 9 This is a graph showing the measurement results of the hair coverage described in the examples of this application.

[0018] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] Description of sequence listing (sequence listing content is provided separately): Seq ID NO.1 shows the amino acid sequence of the extracellular matrix collagen in the examples of this application; Seq ID NO.2 shows the nucleotide sequence of the extracellular matrix collagen in the examples of this application.

[0021] Example 1 Preparation of extracellular matrix collagen 1 Sequence selection Collagen is divided into 29 subtypes. The present application compares the conserved regions of 29 collagens, fuses multiple subtypes of collagen by synthetic biology methods according to the functions of collagen, and utilizes the brewer's yeast expression system, which is endotoxin-free, has high yield, simple culture medium, stable expressed protein, can be secreted to the extracellular space for easy purification, and has the functions of correctly processing, modifying, and reasonably spatially folding heterologous proteins, to heterologously express the target protein, thereby obtaining the extracellular matrix collagen used for scalp care in the present application, the amino acid sequence of which 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.

[0022] Furthermore, based on the properties of the Saccharomyces cerevisiae expression plasmid pYES2 / CT-MFα and the codon preference of the Saccharomyces cerevisiae host, the gene sequence encoding the extracellular matrix collagen was optimized, including reducing the repetition frequency of the repetitive sequence and artificial design, which helped to reduce errors in the translation process and increase the proportion of correctly folded proteins, thereby obtaining the optimized gene sequence of the extracellular matrix collagen, as shown in Seq ID NO.2.

[0023] 2 Construction and screening of recombinant yeast 2.1 Construction of recombinant plasmid According to the gene sequence of extracellular matrix collagen as shown in Seq ID NO.2, General Bio (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 (such as Figure 1 As shown), the recombinant plasmid pYES2 / CT-MFα-ECMCOL was obtained.

[0024] 2.2 Electrotransformation into Saccharomyces cerevisiae INVSc1 competent cells Take 10 μL of recombinant plasmid pYES2 / CT-MFα-ECMCOL and add it to 80 μL of Saccharomyces cerevisiae INVScl competent cells, mix well and transfer to a pre-cooled electroporation cup, and let it stand on ice for 5 minutes; adjust the Bio-Rad electroporation instrument to the fungus mode, and place the electroporation cup on the Bio-Rad electroporation instrument for electroporation transformation; after the electroporation, quickly add 500 μL of pre-cooled 1M sorbitol solution to the electroporation cup, mix well, incubate at 30°C for 1 hour, centrifuge and discard 400 μL-500 μL of supernatant, blow evenly and spread on the SC-U solid plate; incubate the SC-U solid plate at a constant temperature of 30°C until a monoclonal colony grows.

[0025] 2.3 Screening of positive clones Pick the monoclonal colonies grown on the SC-U solid plate and inoculate them into a centrifuge tube containing 500 μL YPD liquid medium. Culture them overnight at 30°C and 180 rpm. Then select 10 monoclonal colonies after overnight culture and extract genomic DNA from them. After PCR amplification of the target gene, positive clones are identified by SDS-PAGE electrophoresis. The electrophoresis identification results are as follows: Figure 2 As shown in the figure (wherein, lane M: 100bp Plus DNA Marker; lane 1: negative control; lanes 2-5: monoclonal colony PCR products), it is basically consistent with the theoretical extracellular matrix collagen band of 1130bp.

[0026] Furthermore, the positive clones that were correctly identified were selected for the next test, thereby obtaining the recombinant yeast engineering strain INVSc1 / pYES2 / CT-MFα-ECMCOL.

[0027] Specifically, the PCR conditions were as follows: 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.

[0028] 3 Inducible expression of recombinant yeast The recombinant yeast INVSc1 / pYES2 / CT-MFα-ECMCOL was selected and inoculated into 20 mL of SC-U selection medium. The culture was shaken at 30°C and 220 rpm overnight. The OD 600nm The absorbance value was calculated, and the corresponding volume of bacterial solution was transferred to 100 mL 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%. After induction, the bacterial solution was centrifuged to collect the induced expression supernatant, and the expression amount of the target protein was analyzed by SDS-PAGE electrophoresis. The results were as follows: Figure 3 As shown (wherein, lane M: protein marker; lane 1: supernatant before induction; lanes 2-4: supernatant of induced expression of different colonies), it can be observed that there is an obvious specific band at around 42 kDa, and the recombinant yeast engineering bacteria INVSc1 / pYES2 / CT-MFα-ECMCOL can produce extracellular matrix collagen of about 42 kDa after induction.

[0029] 4 Purification A cation exchange medium (chromatographic filler is SP Purose 6 High Performance produced by Qianchun, loaded on a GE Akta chromatography system) was used, and the chromatography column was balanced with a phosphate buffer (25Mm NaH2PO4, pH 4.0) until the conductivity value and A280 absorbance value remained unchanged, and the induced expression supernatant was loaded, and the loading flow rate was set to 5mL / min, and the ultraviolet A280 absorbance value was detected. When it increased, the sample was started; after the loading was completed, the cationic chromatography medium was balanced 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 NaH2PO4 buffer containing NaCl (1M) and collected, and the extracellular matrix collagen stock solution was obtained after dialysis. The expression level of the purified protein was analyzed by SDS-PAGE electrophoresis, and the results were as follows: Figure 4 As shown, an obvious specific band around 42 kDa can be observed, which is basically consistent with the predicted molecular weight.

[0030] Example 2 Preparation of finished extracellular matrix collagen composition The extracellular matrix collagen stock solution in Example 1 is mixed with hyaluronic acid to obtain a finished extracellular matrix collagen composition, wherein the final concentration of the extracellular matrix collagen is 4 mg / mL, and the final concentration of the hyaluronic acid is 15%. The finished extracellular matrix collagen composition can be used in hair growth and anti-hair loss products.

[0031] Example 3 Cell adhesion promoting activity detection 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 the pre-dilution, a 2-fold gradient dilution was performed in a 96-well plate, and a total of 8 dilutions were made. 50 μL of the finished sample of the extracellular matrix collagen composition of different dilutions was added to each well. A negative control (without 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 were set up (the three single collagens were diluted to 0.5 μg / mL). After the same 2-fold dilution, different concentrations of single collagen solutions were added to each well; incubation was carried out overnight at 4°C. After the incubation, the liquid in the plate was discarded, and 100 μL of PBS was added to each well for washing for 3 times; after washing, 100 μL of 30 μg / μL bovine serum albumin was added to each well for blocking, and the wells were incubated at 37°C for 1 h; after the incubation, the liquid in the plate was discarded, and 100 μL of PBS was added to each well for washing 3 times, and then the fibroblast suspension was added, and the cell seeding density was 1.0×10 5 cells / mL, 100 μL was inoculated into each well and incubated in an incubator for 5 h; the incubated cell plate was washed with PBS 3 times and the cell adhesion was observed under a 100x microscope. Figure 5 As shown, five points except the edge were selected under a 200x microscope to count the number of adherent cells, and the specific activity was obtained based on the fitting curve of the counting results. The results are shown in Figure 6 shown.

[0032] Depend on Figure 5 It can be seen that only a few cells survived in the negative control wells, and the negative control experiment was established. Figure 6 It can be seen that the extracellular matrix collagen composition of the present application and type I collagen, type III collagen, and type IV collagen all have the activity of promoting fibroblast adhesion, but compared with the three single collagens, the extracellular matrix collagen composition prepared in the present application has better adhesion activity.

[0033] Example 4 Keratinocyte Proliferation Detection The extracellular matrix collagen composition in Example 2 was pre-diluted with PBS buffer to a total protein content of 0.5 μg / mL; the human keratinocyte cell line was cultured with DMEM culture medium containing 10% calf serum at 37°C and 5% CO2, and the cell concentration was controlled to be 1.0×10 4 cells / mL-5.0×10 4 cells / mL, and inoculated in a 96-well plate, shaking continuously during the inoculation process to keep the same number of inoculations in each well, inoculating 100μL in each well, and culturing at 37°C, 5% CO2; after 24h, change to DMEM culture medium with 0.4% calf serum; after culturing for 24h, discard the culture medium, add the pre-diluted extracellular matrix collagen composition product, and set up a negative control group (without any protein) and a single collagen control group (all three collagens were diluted to 0.5μg / mL), each group has 2 replicate wells, 100μL in each well; incubate at 37℃, 5% CO2 for 68h-72h; add 20μL of MTT solution to each well, and incubate at 37℃, 5% CO2 for 5h. The above operations are performed under sterile conditions. After discarding the liquid in the culture plate, add 100μL of dimethyl sulfoxide to each well, fully dissolve and mix, and observe the cell adhesion under a 100x microscope. The results are as follows Figure 7 As shown, at the same time, the absorbance is measured on the microplate reader with 630nm as the reference wavelength and 570nm as the test wavelength, and the measurement results are recorded. The test data are processed by computer program or four-parameter regression calculation method. The results are shown in Figure 8 shown.

[0034] Depend on Figure 7 It can be seen that only a few cells survived in the negative control wells, and the negative control experiment was established. Figure 8 It can be seen that the extracellular matrix collagen composition of the present application and type I collagen, type III collagen, and type IV collagen all have the activity of promoting keratinocyte proliferation, but compared with the three single collagens, the extracellular matrix collagen composition prepared in the present application has a better proliferation effect.

[0035] Example 5 Hair growth promotion experiment After the back hair of C57BL / 6 mice was removed, they were divided into an experimental group and a control group. The experimental group was applied with the extracellular matrix collagen composition in Example 2 on the wound surface of the mice. The control group was divided into 4 groups, which were applied with PBS, type I collagen, type III collagen, and type IV collagen, respectively. The back hair of each mouse was recorded using a digital camera on the 0th day, 4th day, 8th day, and 12th day after treatment, and the hair area was measured using Image J software to calculate the hair coverage. The test results are shown in Figure 2. Fig. 9 shown.

[0036] Depend on Fig. 9 It can be seen that the extracellular matrix collagen composition of the present application has the effect of promoting hair growth as well as 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 better than that of several single collagens, and can achieve a higher hair coverage rate in a short time.

[0037] In summary, the present application compares the conserved regions of 29 collagens, fuses multiple different subtypes of collagen using synthetic biology methods according to the functions of collagen, and optimizes their amino acid sequences and gene sequences to enable them to be efficiently expressed in Saccharomyces cerevisiae. The Saccharomyces cerevisiae expression system is endotoxin-free, has a high yield, a simple culture medium, stable expressed proteins, can be secreted to the extracellular space for easy purification, and has the functions of correctly processing, modifying, and reasonably spatially folding heterologous proteins to heterologously express the target protein, thereby obtaining the extracellular matrix collagen protein for scalp care of the present application, the amino acid sequence of which is shown in Seq ID NO.1, and the gene sequence is shown in Seq ID NO.2. It retains activity that is superior to natural collagen, has low production cost, and stable product quality. It has been verified that compared with a single subtype of collagen, the extracellular matrix collagen after fusion in the present application is more efficiently expressed, has better cell adhesion and cell proliferation activities, higher biological activity, and is more effective in promoting hair growth and scalp repair. It can be used in hair growth and anti-hair loss products. Moreover, since the extracellular matrix collagen is a biological protein, it has the advantage of being easy to decompose and leaving no residue. Compared with ordinary chemical skin care products and hormone drugs, it has the huge advantage of having no side effects.

[0038] The above description is only an optional embodiment of the present application, and does not limit the patent scope of the present application. All equivalent structural transformations made by using the contents of the present application specification and drawings under the inventive concept of the present application, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present 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.

2. The extracellular matrix collagen for scalp care according to claim 1, characterized in that: The gene sequence of the extracellular matrix collagen is shown in Seq ID NO.

2.

3. A method for preparing extracellular matrix collagen for scalp care according to any one of claims 1 to 2, 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.

4. The method for preparing extracellular matrix collagen for scalp care according to claim 3, 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.

5. The method for preparing extracellular matrix collagen for scalp care according to claim 3, 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.

6. The method for preparing extracellular matrix collagen for scalp care according to claim 5, 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.

7. The method for preparing extracellular matrix collagen for scalp care according to claim 6, 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.

8. The method for preparing extracellular matrix collagen for scalp care according to claim 3, 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.

9. The method for preparing extracellular matrix collagen for scalp care according to claim 3, 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.

10. A use of the extracellular matrix collagen for scalp care according to any one of claims 1 to 2, characterized in that: The extracellular matrix collagen is mixed with hyaluronic acid and used in a hair growth and anti-hair loss product.

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