High-activity recombinant XVII type collagen as well as rapid preparation method and application thereof

Abstract

The invention relates to a high-activity recombinant XVII type collagen as well as a rapid preparation method and application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the high-activity recombinant XVII type collagen is as shown in SEQ ID NO. 1, and the nucleotide sequence of the high-activity recombinant XVII type collagen is as shown in SEQ ID NO. 2. The invention also provides a preparation method and application of the recombinant XVII type collagen. The high-activity recombinant XVII type collagen has good biological activity, has good effects of promoting cell adhesion, cell proliferation and cell migration, and can be compounded with other proteins to form raw materials of tissue engineering products, cosmetics, health care products or medicines. The high-activity recombinant XVII type collagen gene is integrated to different positions of a yeast genome through three different vectors, and the copy number of a target gene is increased, so that the expression quantity of the recombinant XVII type collagen is remarkably increased, and the yield of the high-activity recombinant XVII type collagen is effectively increased.

Description

Technical Field

[0001] The invention relates to a highly active recombinant type XVII collagen and a rapid preparation method and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Collagen is a type of fibrous, macromolecular protein with excellent biological functional properties such as biocompatibility and biodegradability. It has become one of the most widely used protein materials in the fields of biomaterials and regenerative medicine. It is widely used in cosmetic filling, drug delivery systems, surgical sutures and tissue engineering scaffolds, and plays a supporting, repairing and anti-aging role. Based on the differences in their protein structure and amino acid composition, human collagen can be divided into 28 types, of which type XVII collagen is basement membrane zone collagen with a total length of 1497 amino acids, including three major structural domains: intracellular, transmembrane and extracellular, with a total of 16 non-triple helical regions and 15 triple helical regions. As a transmembrane protein on the hemidesmosome, it is mainly responsible for tightly combining the epidermis with the dermis to ensure the integrity of the skin structure.

[0003] In addition, type XVII collagen can also play an important role in the repair of skin wounds by affecting the migration, proliferation and differentiation of stem cells. However, the content of type XVII collagen in the human body and animals is extremely low, and it is extremely difficult to extract. Therefore, biotechnology such as genetic engineering is usually used to obtain recombinant type XVII collagen.

[0004] Chinese patent document CN117756926A discloses a recombinant type XVII collagen Pro.C17 and its preparation method and application, which uses Escherichia coli as the expression host. Chinese patent document CN118307663A discloses a recombinant type XVII collagen with a triple helical structure and its application, which uses a single plasmid to transform conventional Pichia pastoris for production, and uses a nickel column for affinity purification, which has a high purification cost and is difficult to scale up in batches.

[0005] At the same time, the yield of type XVII collagen produced by these two preparation methods is relatively low, and the production cost is high. Therefore, it is urgent to develop a new type of recombinant type XVII collagen and a rapid preparation method suitable for industrial production. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a highly active recombinant type XVII collagen and a rapid preparation method and application thereof.

[0007] The technical solution of the present invention is as follows:

[0008] A highly active recombinant type XVII collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The nucleotide sequence of the gene encoding the highly active recombinant type XVII collagen is shown in SEQ ID NO.2.

[0010] A recombinant expression vector is provided, in which the coding gene of the highly active recombinant type XVII collagen is inserted.

[0011] Preferably according to the present invention, the expression vector is pPIC9K, pPICZαA or pGAPCαA.

[0012] A recombinant cell, in which the coding gene or recombinant expression vector of the highly active recombinant type XVII collagen is inserted.

[0013] Preferably according to the present invention, the host cell is protease-deficient Pichia pastoris SMD1163.

[0014] The method for preparing the above-mentioned recombinant type XVII collagen comprises the following steps:

[0015] (1) The highly active recombinant type XVII collagen encoding gene as shown in SEQ ID NO. 2 was connected to the expression vectors pPIC9K, pPICZαA and pGAPCαA, respectively, to obtain the recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P;

[0016] (2) sequentially transforming the recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P obtained in step (1) into Pichia pastoris SMD1163 competent cells to obtain a recombinant strain;

[0017] (3) culturing the recombinant strain obtained in step (2), centrifuging and collecting the supernatant;

[0018] (4) Separating and purifying the supernatant obtained in step (3) to obtain highly active recombinant type XVII collagen.

[0019] Preferably, according to the present invention, in step (1), the expression vector pGAPCαA is constructed according to the following method:

[0020] The pNZ8148 plasmid was used as a template and Cm-F / R as primers to obtain the chloramphenicol resistance gene fragment by PCR amplification; then the expression vector pGAPZαA was double-digested with NcoⅠ and EcoRV endonucleases, and the amplified chloramphenicol resistance gene fragment was connected to the double-digested pGAPZαA vector by homologous recombination to obtain the pGAPCαA vector;

[0021] The sequence of primer Cm-F / R is as follows:

[0022] Cm-F:

[0023] 5′-ACAAGGTGAGGAACTAAACCATGAACTTTAATAAAATTGATTTAGACAATTGG-3′;

[0024] Cm-R: 5′-TGACATAACTAATTACATGATATCTAAAAACCTTCTTCAACTAACGGG-3′.

[0025] Preferably, according to the present invention, in step (4), the separation and purification is specifically:

[0026] The supernatant was first concentrated using a 30 kDa ultrafiltration membrane to collect the permeate; then concentrated using a 1 kDa ultrafiltration membrane to collect the retentate to obtain a protein concentrate; then acetone was added to the protein concentrate under ice bath conditions while stirring to make the final acetone content 40%, the resulting mixed solution was allowed to stand at 4°C overnight, then centrifuged at 4°C, 10000 rpm for 10 min, and the precipitate was collected; the precipitate was re-dissolved with ultrapure water and passed through a cation exchange column, the resulting chromatographic solution was concentrated by ultrafiltration using a 1 kDa membrane, and finally the concentrate was freeze-dried.

[0027] Application of the above-mentioned highly active recombinant type XVII collagen and the highly active recombinant type XVII collagen prepared by the preparation method in any of the following:

[0028] a) Promote cell adhesion;

[0029] b) promote cell proliferation;

[0030] c) promote cell migration;

[0031] d) preparing cosmetics;

[0032] e) Preparation of medical materials.

[0033] The cell proliferation promoting agent can be used as a basic component of a culture fluid to promote cell proliferation, including application in the culture and preservation of in vitro hair follicle stem cells or hair follicle tissues.

[0034] The cell migration promoting agent can be used as a basic component of a culture medium to promote cell migration, including application in the culture and preservation of hair follicle stem cells or hair follicle tissues in vitro.

[0035] The cosmetics include but are not limited to skin care lotion, skin care cream, essence water, facial mask, scalp essence, and shampoo.

[0036] The medical materials include but are not limited to filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, and drug release carriers, wherein the repair materials include but are not limited to bone repair materials, wound dressings, and sutures.

[0037] The beneficial effects of the present invention are:

[0038] 1. The present invention provides a novel highly active recombinant type XVII collagen, which has good biological activity, good cell adhesion, cell proliferation and cell migration promoting effects, and can be compounded with other proteins to become raw materials for tissue engineering products, cosmetics, health products or medicines.

[0039] 2. The present invention integrates the highly active recombinant type XVII collagen gene into different positions of the yeast genome through three different vectors, thereby increasing the copy number of the target gene, thereby significantly increasing the expression level of the recombinant type XVII collagen, and effectively increasing the yield of the highly active recombinant type XVII collagen.

[0040] 3. The present invention optimizes the purification process of recombinant type XVII collagen, improves the purification efficiency of recombinant type XVII collagen, reduces the production cost of recombinant type XVII collagen, increases the production speed of highly active recombinant type XVII collagen, and realizes the rapid preparation of highly active recombinant type XVII collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The SDS-PAGE electrophoresis diagrams are of the supernatant of Pichia pastoris SMD1163 (blank), the supernatant of recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid), and the supernatant of recombinant strain SMD1163-KZG-COL17A1-P (three plasmids).

[0042] Figure 2 This is the plasmid map of the recombinant vector pPIC9K-COL17A1-P.

[0043] Figure 3 This is the plasmid map of the recombinant vector pPICZαA-COL17A1-P.

[0044] Figure 4 This is the plasmid map of the recombinant vector pGAPCαA-COL17A1-P.

[0045] Figure 5 The freeze-dried sponge image and electrophoresis image of the highly active recombinant type XVII collagen COL17A1-P of the present invention;

[0046] In the figure, A is a freeze-dried sponge image of recombinant type XVII collagen COL17A1-P, and B is an electrophoresis image of the freeze-dried sponge image of recombinant type XVII collagen COL17A1-P.

[0047] Figure 6 This is the verification result of the high-activity recombinant type XVII collagen COL17A1-P of the present invention in promoting cell proliferation activity.

[0048] Figure 7 This is the verification result of the cell adhesion promoting activity of the highly active recombinant type XVII collagen COL17A1-P of the present invention.

[0049] Figure 8 This is the verification result of the anti-hair loss activity of the highly active recombinant type XVII collagen COL17A1-P of the present invention. DETAILED DESCRIPTION

[0050] The technical scheme of the present invention is further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. Unless otherwise specified, the reagents and drugs involved in the present invention are all common commercial products.

[0051] The protease-deficient Pichia pastoris SMD1163 used in the present invention is a common commercially available strain, which is sold by Invitrogen.

[0052] The expression vectors pNZ8148, pPIC9K, pPICZαA and pGAPZαA used in the present invention are conventional plasmid expression vectors, which are available from Invitrogen.

[0053] Example 1: Construction of a novel highly active recombinant type XVII collagen COL17A1-P expression vector

[0054] 1. Design of a novel highly active recombinant type XVII collagen COL17A1-P

[0055] The sequence of full-length human type XVII collagen was obtained from the NCBI website, with the accession number KAI4077360.1. The website (www.ebi.ac.uk / interpro) was then used to analyze the sequence of full-length human type XVII collagen, and a new amino acid sequence of highly active recombinant type XVII collagen was designed, named COL17A1-P, and the specific sequence is as follows:

[0056] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGGE

[0057] KGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGP

[0058] MGPPGPKGGPPGPPGPPGPKGDQGQGPPGPPGPQGPKGDKGDPGVPGGQKGEMGTPPGKG

[0059] DRG PAGPPGHPGP PGPRGHKGEK GDKGDQ (SEQ ID NO. 1).

[0060] 2. Construction of recombinant vector

[0061] (1) Construction of pPIC9K-COL17A1-P and pPICZαA-COL17A1-P expression vectors

[0062] BGI was commissioned to artificially synthesize the gene fragment (COL17A1-P) of the highly active recombinant type XVII collagen COL17A1-P of the present invention according to the nucleotide sequence information shown in SEQ ID NO.2. Then, the gene COL17A1-P, expression vector pPIC9K, and pPICZαA were double-digested with EcoRI and NotI enzymes, and then the digested gene COL17A1-P was connected to the two digested expression vectors with T4 DNA ligase to construct the recombinant vector pPIC9K-COL17A1-P (structure as shown in FIG. Figure 2 As shown), pPICZαA-COL17A1-P (structure as Figure 3 shown).

[0063] Among them, double enzyme digestion, ligase ligation and other operations are basic molecular biology experimental operation techniques, and you can refer to the "Molecular Cloning Experiment Guide".

[0064] (2) Construction of pGAPCαA-COL17A1-P expression vector

[0065] The chloramphenicol resistance gene fragment was obtained by PCR amplification using the pNZ8148 plasmid as a template and Cm-F / R as primers. The sequence of primer Cm-F / R is shown in Table 1. The expression vector pGAPZαA was then double-digested with NcoⅠ and EcoRV endonucleases, and the amplified chloramphenicol resistance gene fragment was connected to the double-digested pGAPZαA vector by homologous recombination to obtain the pGAPCαA vector.

[0066] Table 1. Primer sequences

[0067] Primer name Sequence 5′-3′ Cm-F ACAAGGTGAGGAACTAAACCATGAACTTTAATAAAATTGATTTAGACAATTGG Cm-R TGACATAACTAATTACATGATATCTAAAAACCTTCTTCAACTAACGGG

[0068] Then, the gene COL17A1-P and the expression vector pGAPCαA were double-digested with EcoRI and NotI enzymes, respectively, and then the digested gene COL17A1-P and the digested expression vector pGAPCαA were ligated with T4 DNA ligase to construct the recombinant vector pGAPCαA-COL17A1-P (structure as shown in Figure 4 shown).

[0069] Among them, PCR amplification, double enzyme digestion, homologous recombination ligation and other operations are basic molecular biology experimental operation techniques, and you can refer to the "Molecular Cloning Experiment Guide".

[0070] Example 2: Construction of recombinant strains

[0071] 1. Construction of recombinant strain SMD1163-K-COL17A1-P

[0072] The recombinant vector pPIC9K-COL17A1-P described in Example 1 was digested with SalI, and the digested plasmid was recovered and electroporated into protease-deficient Pichia pastoris SMD1163 competent cells, and then spread on MD solid medium and inverted cultured at 30°C for 4 days. The grown single colonies were transferred to YPD solid medium containing 1 mg / mL or 4 mg / mL G418 for screening, and the single colonies grown on YPD solid medium containing 4 mg / mL G418 were inoculated into 50 mL YPD liquid medium and cultured overnight at 30°C and 220 rpm to obtain recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid).

[0073] MD solid medium formula: 13.4g / L yeast basic nitrogen source YNB, 0.4mg / L biotin, 20g / L glucose.

[0074] YPD liquid culture medium formula: 10g / L yeast extract powder, 20g / L peptone, 20g / L glucose.

[0075] 2. Construction of recombinant strain SMD1163-KZ-COL17A1-P

[0076] Take 1 mL of the single-plasmid recombinant Pichia pastoris SMD1163-K-COL17A1-P bacterial solution and add it to 50 mL of fresh YPD liquid medium. Incubate at 30°C and 220 rpm in a shaking incubator until the OD 600To 1.3, let stand on ice for 15 min, centrifuge at 4°C, 1500g for 5 min, discard the supernatant, then add 50 mL of pre-cooled sterile water to resuspend the cells, centrifuge at 4°C, 1500g for 5 min, discard the supernatant, then add 25 mL of pre-cooled sterile water to resuspend the cells, centrifuge at 4°C, 1500g for 5 min, discard the supernatant, then add 10 mL of pre-cooled 1 mol / L sorbitol to resuspend the cells, centrifuge at 4°C, 1500g for 5 min, discard the supernatant, add 1 mL of pre-cooled 1 mol / L sorbitol to resuspend the cells, and distribute them into sterile centrifuge tubes to obtain Pichia pastoris SMD1163-K-COL17A1-P competent cells, and store them in a -80°C refrigerator for later use.

[0077] The recombinant vector pPICZαA-COL17A1-P described in Example 1 was digested with PmeI, and the digested plasmid was recovered and then coated on a YPDZ solid medium containing 100 μg / mL bleomycin, and inverted and cultured at 30°C for 48 h. The grown single colonies were transferred to a YPDZ solid medium containing 400 μg / mL bleomycin for screening, and the grown single colonies were inoculated into 50 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm in a shaking incubator to obtain the dual-plasmid recombinant Pichia pastoris SMD1163-KZ-COL17A1-P.

[0078] Preparation method of YPDZ solid culture medium: Add bleomycin at a final concentration of 100 μg / mL and 400 μg / mL to YPD culture medium respectively.

[0079] 3. Construction of SMD1163-KZG-COL17A1-P recombinant strain

[0080] Prepare the dual-plasmid recombinant Pichia pastoris SMD1163-KZ-COL17A1-P competent cells according to the method obtained in step 2.

[0081] The recombinant vector pGAPCαA-COL17A1-P described in Example 1 was digested with BspHⅠ, and the digested plasmid was recovered and electroporated into the dual-plasmid recombinant Pichia pastoris SMD1163-KZ-COL17A1-P competent cells, followed by coating with YPDC solid culture medium containing 25 μg / mL chloramphenicol, inverted culture at 30°C for 3 days, and the grown single colony was inoculated into 50 mL YPD liquid culture medium, and cultured in a shaking incubator at 30°C and 220 rpm overnight to obtain the recombinant strain SMD1163-KZG-COL17A1-P (three plasmids).

[0082] YPDC medium preparation method: add chloramphenicol with a final concentration of 25 μg / mL to the YPD medium.

[0083] Example 3: Fermentation of recombinant strains in fermenter

[0084] 1. The recombinant strain SMD1163-KZG-COL17A1-P constructed in Example 2 was inoculated into YPD liquid culture medium at a volume ratio of 5%, and cultured on a shaking table at 30° C. and 220 rpm for 20 h to obtain seed solution.

[0085] 2. The seed solution obtained in step 1 was inoculated into a 10L fully automatic fermenter at a volume ratio of 5%. At the beginning of the reaction, the fermenter contained 4L of BSM Plus liquid culture medium, the temperature was controlled at 30°C, the pH was 5.0, the stirring speed was 200rpm, the ventilation volume was 10L / min, and the dissolved oxygen was maintained above 25%. After culturing for 18h, the initial glycerol was consumed, the dissolved oxygen recovered, and 50% (w / V) glycerol (containing 12mL / L PTM1) was added at a rate of 75g / h. During this period, the dissolved oxygen was controlled to be greater than 25%. When the wet weight of the bacteria reached 180g / L, the glycerol addition was stopped, the fermentation temperature was lowered to 25°C, the starvation culture was carried out for 60min, and then 100% methanol (containing 12mL / L PTM1, the specific methanol flow rate is: first add 12 g / h for 2 h, then add 18 g / h for 2 h, then add 24 g / h for 2 h, and finally add 33 g / h until the induced fermentation is completed). The timing starts after the methanol is added. The induced fermentation culture is stopped for 96 h (the dissolved oxygen is controlled to be greater than 25% during the induced fermentation process) to obtain a fermentation broth. After the fermentation is completed, the fermentation broth is centrifuged at 4°C, 8000 rpm for 10 min, and the supernatant is collected.

[0086] At the same time, the protease-deficient Pichia pastoris SMD1163 and the recombinant Pichia pastoris SMD1163-K-COL17A1-P constructed in Example 2 were cultured in the same manner and the supernatant was collected. Then, the supernatant of Pichia pastoris SMD1163 (blank), the supernatant of recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid), and the supernatant of recombinant strain SMD1163-KZG-COL17A1-P (three plasmids) were subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 1 shown.

[0087] Depend on Figure 1 It can be seen that the supernatant of Pichia pastoris SMD1163 (blank) does not contain recombinant type XVII collagen COL17A1-P, while the supernatant of recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid) and the supernatant of recombinant strain SMD1163-KZG-COL17A1-P (three plasmids) contain recombinant type XVII collagen COL17A1-P with a molecular weight of approximately 22 kDa, indicating that the heterologous expression of recombinant type XVII collagen COL17A1-P is successful.

[0088] In this embodiment, the yield of recombinant type XVII collagen COL17A1-P in the supernatant of recombinant Pichia pastoris SMD1163-K-COL17A1-P (single plasmid) is 5.5 g / L, and the yield of recombinant type XVII collagen COL17A1-P in the supernatant of recombinant strain SMD1163-KZG-COL17A1-P (three plasmids) is 13 g / L. This shows that the present invention integrates the highly active recombinant type XVII collagen gene into different positions of the yeast genome through three different vectors, increases the copy number of the target gene, thereby significantly increasing the expression amount of recombinant type XVII collagen, and effectively increasing the yield of recombinant type XVII collagen.

[0089] BSM Plus liquid culture medium formula: calcium sulfate dihydrate 0.47g / L, potassium sulfate 9.1g / L, magnesium sulfate heptahydrate 7.5g / L, potassium hydroxide 2.1g / L, glycerol 40g / L, phosphoric acid 13.4ml / L, yeast extract powder 10g / L, L-histidine 0.5g / L, defoaming agent 0.5g / L.

[0090] Example 4: Rapid purification method of recombinant type XVII collagen COL17A1-P

[0091] The fermentation broth of recombinant Pichia pastoris SMD1163-K-COL17A1-P prepared in Example 3 was collected, and the fermentation broth was centrifuged at 10,000 r / m for 30 min to obtain a supernatant. The supernatant was concentrated using a 30 kDa ultrafiltration membrane, and the permeate was collected. Then, the supernatant was concentrated using a 1 kDa ultrafiltration membrane, and the retentate was collected to obtain a protein concentrate. Then, acetone was added to the protein concentrate under ice bath conditions while stirring to make the final acetone content 40%. The resulting mixed solution was allowed to stand overnight at 4°C, and then centrifuged at 4°C and 10,000 rpm for 10 min to collect the precipitate. The precipitate was re-dissolved with ultrapure water and passed through a cation exchange column. The resulting chromatographic solution was concentrated by ultrafiltration using a 1 kDa membrane, and finally the concentrate was freeze-dried to obtain purified recombinant type XVII collagen COL17A1-P. The freeze-dried sponge and protein electrophoresis diagram of the recombinant type XVII collagen COL17A1-P are shown in the figure. Figure 5 shown.

[0092] Depend on Figure 5 It can be seen that the molecular weight of the protein in the electrophoresis diagram is about 22 kDa, indicating that the recombinant type XVII collagen COL17A1-P was successfully separated and purified in this example.

[0093] Example 5. Activity detection of recombinant type XVII collagen COL17A1-P

[0094] 1. Recombinant type XVII collagen COL17A1-P promotes cell proliferation activity

[0095] Human immortalized keratinocytes HaCaT were used as the research object. First, human immortalized keratinocytes HaCaT in the exponential growth phase were collected and counted. 4 / well, spread the cells in a 96-well plate, set up 5 duplicate wells; after culturing for 24 hours, add 1 mg / mL of the recombinant type XVII collagen COL17A1-P prepared in Example 4, continue culturing for 48 hours, aspirate the original culture medium and wash once with PBS, add fresh culture medium, and add 10 mL of CCK8 to each well (CCK8 reagent detection method is a highly sensitive, non-radioactive colorimetric detection method used to determine the number of live cells in cell proliferation or toxicity experiments). After incubation at 37°C for 1 hour, use a multifunctional microplate reader to detect the absorbance of each well at a wavelength of 450nm, and statistically analyze the cell viability of each group. Commercially available bovine serum albumin BSA and commercially available recombinant type XVII collagen were used as reference controls. The experimental results are as follows Figure 6 shown.

[0096] Depend on Figure 6 It can be seen that the recombinant type XVII collagen COL17A1-P of the present invention has a tendency to promote the activity of HaCaT cells within the concentration gradient of 0.25 to 4 mg / mL, has no toxic effect, and the difference is statistically significant. This shows that the recombinant type XVII collagen COL17A1-P of the present invention is better than the control group BSA and the commercially available recombinant type XVII collagen in promoting the proliferation of HaCaT cells, and is a highly active recombinant type XVII collagen COL17A1-P.

[0097] 2. Cell adhesion activity of recombinant type XVII collagen COL17A1-P

[0098] Take the purified lyophilized product of recombinant type XVII collagen COL17A1-P prepared in Example 4, the control commercial bovine serum albumin (BSA) and the commercial recombinant type XVII collagen, dissolve them in pure water, and determine the protein concentration using the UV protein quantitative empirical formula: C (mg / mL) = 0.144 × (A215-A225), and then dilute to 0.5 mg / mL with PBS buffer (pH = 7.4). Add 100 μL of each protein solution to be tested to a 96-well cell culture plate, and use a blank PBS buffer as a control. Let it stand at room temperature for 60 minutes; then add 10 5 NIH / 3T3 cells in good culture state were incubated at 37°C and 5% CO2 for 60 minutes. The cells in the wells were washed 4 times with PBS buffer. The absorbance value at OD = 492nm was measured using the LDH detection kit to detect the cell migration activity. The results are shown in Figure 7As shown in the figure, the absorbance at OD = 492 nm corresponds to the cell adhesion activity of the collagen sample: the higher the absorbance, the more cells adhere to the protein and the higher the adhesion activity.

[0099] Depend on Figure 7 It can be seen that the cell adhesion activity of the recombinant type XVII collagen COL17A1-P of the present invention is better than that of BSA and commercially available recombinant type XVII collagen, indicating that it can help cells adhere to the wall or extracellular matrix in a short period of time, and is more conducive to building a better extracellular environment. It is a highly active recombinant type XVII collagen COL17A1-P.

[0100] 3. Recombinant type XVII collagen COL17A1-P prevents hair loss

[0101] The recombinant type XVII collagen COL17A1-P concentrate prepared in Example 4 (Group A), the commercially available recombinant type XVII collagen essence matrix reference substance (Group B), and Mandiminoxidil tincture (Group C, minoxidil tincture is a peripheral vasodilator approved by the State Food and Drug Administration, which can stimulate hair growth in patients with male pattern baldness and alopecia areata when used topically.) were used as test samples, and the anti-hair loss effect was tested on humans.

[0102] Test groups: Group A, 11 people were enrolled, and a total of 11 valid samples were completed. Among them, there were 6 females and 5 males, with an age range of 34 to 50 years old, and an average age of 41.7±4.9 years old. Group B, 11 people were enrolled, and a total of 11 valid samples were completed. Among them, there were 6 females and 5 males, with an age range of 24 to 54 years old, and an average age of 41.8±8.1 years old. Group C, 11 people were enrolled, and a total of 11 valid samples were completed. Among them, there were 7 females and 4 males, with an age range of 26 to 49 years old, and an average age of 39.9±6.8 years old. The subjects' hair length was between 5 and 40 cm, and they had problems with a lot of hair loss and mild thinning hair. The hair loss count according to the 60-time combing method was greater than 10, and it was still greater than 10 after a 2-week washing period, which met the project requirements.

[0103] Test process: During the test, recombinant type XVII collagen essence and recombinant type XVII collagen essence matrix reference substance: 1 bottle per day, use 2 to 3 times a day, tear open the bottle mouth in the direction of tearing the aluminum seal, install the matching dropper, separate the hair, evenly apply it on the scalp, and gently massage to promote its absorption. No need to wash after use. Mandiminoxidil tincture: once a day, 1mL each time (about 7 sprays), apply to the affected area of ​​the head, start applying from the center of the affected area, and massage with your hands for 3 to 5 minutes. Regardless of the size of the affected area, this dose is used, and the total amount per day does not exceed 2mL. The test results are as follows: Figure 8 shown.

[0104] Depend on Figure 8 It can be seen that with the extension of the use time, the amount of hair loss in the experimental group A decreased significantly, indicating that the recombinant type XVII collagen COL17A1-P of the present invention has the effect of slowing down the reduction of hair quantity and preventing hair loss.

Claims

1. A highly active recombinant type XVII collagen, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the highly active recombinant type XVII collagen according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The gene encoding the highly active recombinant type XVII collagen according to claim 2 is inserted into the expression vector.

4. The recombinant expression vector according to claim 3, characterized in that: The expression vector is pPIC9K, pPICZαA or pGAPCαA.

5. A recombinant cell, characterized in that The gene encoding the highly active recombinant type XVII collagen according to claim 2 or the recombinant expression vector according to claim 3 is inserted into the host cell.

6. The recombinant cell according to claim 3, characterized in that The host cell is a protease-deficient Pichia pastoris SMD1163.

7. The method for preparing the highly active recombinant type XVII collagen according to claim 1, characterized in that: The steps include: (1) The highly active recombinant type XVII collagen encoding gene as shown in SEQ ID NO. 2 was connected to the expression vectors pPIC9K, pPICZαA and pGAPCαA, respectively, to obtain the recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P; (2) sequentially transforming the recombinant vectors pPIC9K-COL17A1-P, pPICZαA-COL17A1-P and pGAPCαA-COL17A1-P obtained in step (1) into Pichia pastoris SMD1163 competent cells to obtain a recombinant strain; (3) culturing the recombinant strain obtained in step (2), centrifuging and collecting the supernatant; (4) Separating and purifying the supernatant obtained in step (3) to obtain highly active recombinant type XVII collagen.

8. The preparation method according to claim 7, characterized in that: In step (4), the expression vector pGAPCαA is constructed as follows: Using pNZ8148 plasmid as template, the chloramphenicol resistance gene fragment was obtained by PCR amplification, and then the expression vector pGAPZαA was double-digested with NcoⅠ and EcoRV endonucleases. The amplified chloramphenicol resistance gene fragment was connected to the double-digested pGAPZαA vector by homologous recombination to obtain the pGAPCαA vector.

9. The preparation method according to claim 7, characterized in that: In step (4), the separation and purification specifically includes: The supernatant was first concentrated using a 30 kDa ultrafiltration membrane to collect the permeate; then concentrated using a 1 kDa ultrafiltration membrane to collect the retentate to obtain a protein concentrate; then acetone was added to the protein concentrate under ice bath conditions while stirring to make the final acetone content 40%, the resulting mixed solution was allowed to stand at 4°C overnight, then centrifuged at 4°C, 10000 rpm for 10 min, and the precipitate was collected; the precipitate was re-dissolved with ultrapure water and passed through a cation exchange column, the resulting chromatographic solution was concentrated by ultrafiltration using a 1 kDa membrane, and finally the concentrate was freeze-dried.

10. Use of the highly active recombinant type XVII collagen according to claim 1 or the highly active recombinant type XVII collagen prepared by the preparation method according to claim 7 in any of the following: a) Promote cell adhesion; b) promote cell proliferation; c) promote cell migration; d) preparing cosmetics; e) Preparation of medical materials.

Images