Use of recombinant calnexin for hair growth, hair development, hair protection or hair loss prevention and related products

Abstract

The application discloses purposes and related products of recombinant calreticulin (CRT) in hair growth, hair growth, hair protection or hair loss prevention. The recombinant protein is derived from full-length calreticulin or a fragment thereof, and has the functions of treating hair loss and improving hair by injection or external application. Therefore, calreticulin is expected to be developed into an effective method and means for treating hair loss and improving hair.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, specifically to the use of recombinant calreticulin in hair growth, hair development, hair protection, or hair loss prevention, and related products. Technical Background

[0002] Hair loss is a common clinical condition, and its causes are not fully understood. It may be related to immune status, genetic factors, hormone levels, local inflammatory responses, neurological factors, and environmental factors. Common types of hair loss include androgenetic alopecia (AGA), alopecia areata, telogen effluvium, and age-related hair loss, with AGA being the most prevalent. Literature reports that the prevalence of AGA is 30% in 30-year-old Caucasian men, as high as 50% in 50-year-old men, and 80% and 53% in men and women over 70 years of age, respectively. Literature also reports that the prevalence of AGA in China is 21.3% in men and 6.0% in women. Currently, only finasteride and minoxidil are approved by the US FDA for the treatment of hair loss. However, the clinical efficacy of these two drugs is unsatisfactory, and they have certain side effects. Therefore, there is an urgent need for safe and effective new drugs and treatment methods.

[0003] Calreticulin (CRT) was first discovered in the endoplasmic reticulum (ER) and is highly conserved throughout evolution. Belonging to the heat shock protein family, CRT is primarily located in the ER and possesses a variety of biological functions. CRT is a Ca2+-binding protein; the human CRT has a molecular weight of approximately 46 kDa and consists of three domains: N, P, and C. The junction between the N and P domains is a crucial region for this protein to perform its molecular chaperone function. The P domain has a high affinity for calcium ions, but the amount bound is relatively small; conversely, the C domain has a low affinity for calcium ions but a large capacity, and is considered an intracellular "calcium reservoir." Recent studies have shown that CRT also exists outside the ER and plays important biological functions, such as participating in antigen processing and presentation in adaptive immunity, anti-tumor immune responses, mediating phagocytosis of apoptotic cells, cell adhesion and migration, and the proliferation and activation of immune cells. Later studies revealed that calreticulin (CRT) also exists in the cellular matrix and on the membranes of almost all immune cells, including monocytes / macrophages, neutrophils, T cells, and dendritic cells, playing a crucial role in cell activation and the elimination of apoptotic and tumor cells. Calcium ions are important intracellular second messengers, and changes in intracellular calcium ion concentration significantly affect many cellular functions, including the secretion of various cellular bioactive substances, muscle contraction and relaxation, protein modification, and gene expression. The endoplasmic reticulum is a vital site for the folding, modification, and assembly of most proteins. During protein synthesis, misfolding is inevitable. In such cases, calreticulin acts as a molecular chaperone, repairing proteins under the action of various enzymes. CRT can also bind to calrenin to jointly repair abnormally synthesized glycoproteins.

[0004] Currently, no literature or related reports have revealed the application of CRT in hair growth and hair improvement. Summary of the Invention

[0005] To address the aforementioned technical limitations, this invention proposes a calreticulin, a DNA sequence encoding the protein, a vector or plasmid containing the DNA sequence, a host cell, a method for preparing the protein using genetic engineering, and the application of the protein in hair growth, hair development, hair loss prevention, and promoting or protecting hair growth, thus overcoming the deficiencies and defects mentioned in the background art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The purpose of this invention is to provide the calreticulin involved, the DNA sequence encoding the protein, the vector or plasmid containing the DNA sequence, the host cell, the method for preparing the protein by genetic engineering, and the application of the protein in hair growth, hair development, hair loss prevention, and promoting or protecting hair growth.

[0008] To achieve the above objectives, the present invention employs the following technical solution:

[0009] This invention discloses the use of calreticulin (CRT) in the preparation of products for hair growth, hair development, hair protection, or hair loss prevention.

[0010] Preferably, the calreticulin CRT is a recombinantly expressed full-length CRT protein (1-400 aa), rh-CRT-1, whose amino acid sequence is shown in SEQ ID NO:1, or a protein with at least 85% similarity to the amino acid sequence shown in SEQ ID NO:1 and having similar functions.

[0011] The aforementioned proteins include CRT-1 homologous sequences with similarities of 85%, 90%, 95%, and 99% (i.e., 340, 360, 380, and 396 aa, respectively, identical to the full-length CRT protein).

[0012] Preferably, the calreticulin CRT is a protein containing a recombinantly expressed CRT fragment (fragment 1-349aa), which is rh-CRT-2, and its amino acid sequence is shown in SEQ ID NO:9, or a protein that has at least 85% similarity to the amino acid sequence shown in SEQ ID NO:9 and has similar functions.

[0013] The aforementioned proteins include CRT-2 homologous sequences with similarities of approximately 86%, 89%, 95%, and 99% (i.e., 300, 310, 330, and 345 aa, respectively, identical to the 349aa CRT fragment protein).

[0014] Preferably, the calreticulin CRT is a protein containing a recombinantly expressed CRT fragment (fragment 1-291aa), which is rh-CRT-3, and its amino acid sequence is shown in SEQ ID NO:12, or a protein that has at least 85% similarity to the amino acid sequence shown in SEQ ID NO:12 and has similar functions.

[0015] The aforementioned proteins include CRT-3 homologous sequences with similarities of approximately 86%, 91%, 96%, and 99% (i.e., 250, 265, 280, and 288 aa, respectively, identical to the 291aa CRT fragment protein).

[0016] This invention discloses a recombinant protein related to hair growth, hair development, hair protection, or hair loss prevention. The recombinant protein is rh-CRT-1, rh-CRT-2, or rh-CRT-3. The amino acid sequence of rh-CRT-1 is shown in SEQ ID NO:1, the amino acid sequence of rh-CRT-2 is shown in SEQ ID NO:9, and the amino acid sequence of rh-CRT-3 is shown in SEQ ID NO:12.

[0017] Furthermore, the genes encoding the recombinant proteins related to hair growth, hair development, hair protection, and hair loss prevention mentioned above have the following nucleotide sequences: rh-CRT-1 as shown in SEQ ID NO:2, rh-CRT-2 as shown in SEQ ID NO:8, and rh-CRT-3 as shown in SEQ ID NO:11.

[0018] The present invention also discloses a plasmid containing the genes of the recombinant proteins mentioned above that are related to hair growth, hair development, hair protection or hair loss prevention.

[0019] The present invention also discloses a carrier that uses the genes of the recombinant proteins mentioned above that are related to hair growth, hair development, hair protection or hair loss prevention as active ingredients.

[0020] This invention discloses the application of the above-mentioned recombinant proteins, encoding genes, plasmids or vectors in the preparation of drugs or medical device products for hair growth, hair development, hair protection or hair loss prevention.

[0021] Preferably, the drug is a drug used to improve the condition of the hair shaft and hair cuticle.

[0022] Preferably, the drug is a drug for increasing the number of hair follicles in the skin.

[0023] Preferably, the drug is a drug that promotes hair growth by reducing male hormone levels.

[0024] More preferably, the male hormone is dihydrotestosterone.

[0025] The present invention also discloses a product with functions of hair growth, hair development, hair protection or hair loss prevention, wherein the active ingredients are the above-mentioned recombinant protein, gene, plasmid or vector.

[0026] The product has at least one of the following uses:

[0027] a) Improves hair smoothness;

[0028] b) Increase hair volume;

[0029] c) Promotes hair growth;

[0030] The product is a drug, additive, or active ingredient.

[0031] The present invention also discloses a drug with functions of promoting hair growth, nourishing hair, protecting hair or preventing hair loss, which is composed of the above-mentioned recombinant protein, gene, plasmid or vector and pharmaceutically additive excipients.

[0032] The drug also contains a combination drug formulation, preferably minoxidil.

[0033] Preferably, the excipients include one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, and lubricant.

[0034] Preferably, the drug can be introduced into body tissues by injection, spraying, penetration, absorption, and physical or chemical mediated methods; or it can be introduced into the body after being mixed or encapsulated with other substances.

[0035] Preferably, the drug is a topical preparation.

[0036] The present invention also discloses the application of calreticulin CRT in the preparation of pharmaceutical or medical device products for the treatment of acne, pimples, or alopecia areata.

[0037] Preferably, the calreticulin CRT is a recombinantly expressed full-length CRT protein with the amino acid sequence shown in SEQ ID NO:1, or a protein with at least 85% similarity to the amino acid sequence shown in SEQ ID NO:1 and having similar functions.

[0038] Preferably, the calreticulin CRT is a protein containing a recombinantly expressed CRT fragment, the amino acid sequence of which is shown in SEQ ID NO:9, or a protein that has at least 85% similarity to the amino acid sequence shown in SEQ ID NO:9 and has similar functions.

[0039] Preferably, the calreticulin CRT is a protein containing a recombinantly expressed CRT fragment, the amino acid sequence of which is shown in SEQ ID NO:12, or a protein that has at least 85% similarity to the amino acid sequence shown in SEQ ID NO:12 and has similar functions.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This invention discloses for the first time the uses of calreticulin (CRT) in hair growth, hair development, hair protection, and hair loss prevention. Furthermore, this invention discloses for the first time a recombinant protein possessing the aforementioned functions of hair growth, hair development, hair protection, or hair loss prevention, and clarifies the preparation method and application of this recombinant protein. This protein is derived from calreticulin or fragments thereof, and proteins with a sequence similarity of 95%, 90%, or 85% to the aforementioned recombinant protein all possess the functions of hair growth, hair development, hair protection, or hair loss prevention. Through thorough experimental verification, this invention demonstrates that the recombinant protein disclosed in this invention, when injected or applied topically, has the function of treating hair loss and improving hair quality. Therefore, CRT protein holds promise for development as an effective method and means of treating hair loss and improving hair quality. Attached Figure Description

[0042] Figure 1 The pPICZαA-rh-CRT-1 plasmid map;

[0043] Figure 2 Electrophoresis results of pPICZαA-rh-CRT-1 plasmid digestion (Xho I / Not I); molecular weight standard unit: bp;

[0044] Figure 3 Image of protein rh-CRT-1 SDS-PAGE electrophoresis;

[0045] Figure 4 Image of pET42a-rhCRT-2 plasmid;

[0046] Figure 5 Electrophoresis results of pET42a-rhCRT-2 plasmid digestion (Nde I / Xho I); molecular weight standard unit: bp;

[0047] Figure 6 Image of protein rh-CRT-2 SDS-PAGE electrophoresis;

[0048] Figure 7 Image of pET42a-rhCRT-3 plasmid;

[0049] Figure 8 Electrophoresis results of pET42a-rhCRT-3 plasmid digestion (Nde I / Xho I); molecular weight standard unit: bp;

[0050] Figure 9 Image of protein rh-CRT-3 SDS-PAGE electrophoresis;

[0051] Figure 10 This serves as the normal control group (observed under a dermoscope).

[0052] Figure 11 For the model group (dermoscopy observation);

[0053] Figure 12 Group A (dermoscopy observation);

[0054] Figure 13 Group B (observed under a dermoscope);

[0055] Figure 14 Positive result for the opposite eye group – minoxidil (dermoscopy observation);

[0056] Figure 15 The positive control group consisted of finasteride (observed under dermoscopy).

[0057] Figure 16 This is the normal control group (observed under a scanning microscope);

[0058] Figure 17 For the model group (observed under a scanning microscope);

[0059] Figure 18 Group A (observed under a scanning microscope);

[0060] Figure 19 Group B (observed under a scanning microscope);

[0061] Figure 20 The positive control group consisted of minoxidil (observed under a scanning microscope).

[0062] Figure 21 The positive control group consisted of finasteride (observed under a scanning microscope).

[0063] Figure 22 Hair growth in each group of mice

[0064] Figure 23 To observe the condition of mice in different groups using a dermoscopy;

[0065] Figure 24 The results of colorimetric assays for each group of mice are shown below.

[0066] Figure 25 Scanning electron microscope images of each experimental group;

[0067] Figure 26 The effects of each experimental group on the number of hair follicles in mice with androgenetic alopecia; (a) pathological sections of hair follicles in experimental groups C and D; (b) the effects of experimental groups C and D on the number of hair follicles in mice with androgenetic alopecia.

[0068] Figure 27 The effects of experimental groups C and D on serum and skin tissue hormones in androgenic alopecia mice were investigated.

[0069] Figure 28This is a comparison of the statistical results of the time and hair growth score of different drug administration groups in the third experiment described in an embodiment of the present invention.

[0070] Figure 29 This is a comparison of the statistical results of the time and hair growth score of different drug administration groups in the fourth experiment described in an embodiment of the present invention. Detailed Implementation

[0071] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated herein.

[0073] To further understand the present invention, the present invention will be described in detail below with reference to the preferred embodiments.

[0074] Example 1

[0075] 1. Expression and purification of recombinant human calreticulin (rh-CRT)

[0076] The human calreticulin gene (CRT, GenBank: M84739.1) is 1958 bp and encodes 417 amino acids, of which amino acids 1-17 are the signal peptide. The sequence of the mature polypeptide (amino acids 18-417) is shown in SEQ ID NO:1; the gene sequence of the mature polypeptide is shown in SEQ ID NO:2.

[0077] 1.1 Cloning and Expression of rh-CRT-1

[0078] Using the fully synthesized SEQ ID NO:2 gene as a template, and pFcrt-1 / pRcrt-1 as upstream and downstream primers, the gene was amplified by PCR. An XhoI restriction site was introduced upstream, and a his-tag (his-his-his-his-his-his) sequence and a NotI restriction site were introduced downstream.

[0079] pFcrt-1 primers:

[0080] 5'-CTCTCTCGAGAAAAGAGAGCCTGCCGTCTACTTCAAG-3' (SEQ ID NO: 3);

[0081] pRcrt-1 primer: 5'-GTAAGCGGCCGCCTAATGATGATGATGATGATGCAGCTCGTCCTTGGCCTG-3' (SEQ ID NO: 4);

[0082] Polymerase chain reaction (PCR) was performed using Phusion, a high-fidelity DNA polymerase from Thermo Scientific, following a 30-cycle sequence of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, followed by a 10-minute incubation at 72°C. The resulting PCR product (approximately 1250 bp) was purified, digested with XhoI / NotI, and inserted into the plasmid pPICZαA (Invitrogen). This plasmid was then transformed into competent *E. coli* TOP10 cells, and positive clones were selected on LB medium containing the antibiotic Zeocin (Invitrogen). The plasmid was extracted, sequenced, and confirmed to be correct. The plasmid was named pPICZαA-rh-CRT-1, and its image is shown below. Figure 1 As shown, the enzyme digestion results are as follows: Figure 2 As shown. pPICZαA-rh-CRT-1, linearized by SacI digestion, was transformed into Pichia pastoris GS115 via electroporation. Positive clones were selected on YPD plates containing the antibiotic Zeocin (100 μg / ml). Twenty positive clones were selected and inoculated into small Erlenmeyer flasks containing 50 ml of BMGY medium. The flasks were incubated at 30°C and 200 RPM until the OD600 reached approximately 1.5. The culture was centrifuged to collect the cells, which were resuspended in 100 ml of BMGY medium and transferred to large Erlenmeyer flasks containing 100 ml of BMGY medium. Incubation was continued at 30°C and 200 RPM. Expression was induced using 0.5% methanol. At 0 h, 24 h, 48 h, 72 h, and 96 h, 1.0 ml of culture medium was collected, centrifuged, and the supernatant was analyzed by SDS-PAGE to determine the target protein expression level. Clones with high expression levels were selected and fermented in a 5 L fermenter for 72 h. The supernatant was collected by centrifugation.

[0083] The culture medium formulation is as follows:

[0084] LB liquid culture medium formula: 10.0g peptone, 5.0g yeast extract, 5.0g sodium chloride, add purified water to 1.0L, adjust the pH to 7.2, dispense into Erlenmeyer flasks, and autoclave at 121℃ for 20 minutes.

[0085] LB solid culture medium formula: 10.0g peptone, 5.0g yeast extract, 5.0g sodium chloride, 15.0g agar powder, add purified water to 1.0L, adjust the pH to 7.2, dispense into Erlenmeyer flasks, and autoclave at 121℃ for 20 minutes.

[0086] YPD medium formula: 20.0g peptone, 10.0g yeast extract, 20.0g glucose, 15.0g agar powder, add purified water to 1.0L, dispense into Erlenmeyer flasks, and autoclave at 115℃ for 20 minutes.

[0087] BMGY culture medium formula: 20.0g peptone, 10.0g yeast extract, 11.8g potassium dihydrogen phosphate, 3.0g dipotassium hydrogen phosphate trihydrate, 10.0mL glycerol, add 900mL purified water, autoclave at 115℃ for 20 minutes. After cooling, add 100mL of sterile filtered 13.4% YNB solution and 2.0mL of sterile filtered 0.02% biotin solution.

[0088] BMMY culture medium formula: 20.0g peptone, 10.0g yeast extract, 11.8g potassium dihydrogen phosphate, 3.0g dipotassium hydrogen phosphate trihydrate, add 800mL purified water, autoclave at 115℃ for 20 minutes. After cooling, add 100mL of sterile filtered 13.4% YNB solution, 2.0mL of sterile filtered 0.02% biotin solution, and 5.0mL of 100mL 5.0% methanol solution.

[0089] 1.2 Purification of rh-CRT-1 protein

[0090] Take 1.0 L of the expression supernatant from the previous step, dilute it with 1.5 L of 20 mM pH 7.5 Tris-HCl buffer, and load it onto an AKTA purifier 100 system. Use a GE XK16 / 20 column and GE Nisepharose 6FF 20 mL packing material. Wash with 20 mM pH 7.5 Tris-HCl buffer containing 100 mM imidazole, elute with 25 mM EDTA solution, and collect the protein peak. Concentrate the protein peak using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa, load it onto an AKTA purifier 100 system, using a GE XK16 / 100 column and GE Sephactyl S-200 packing material. Pack the column to a height of 95 cm, elute with 10 mM pH 7.2 PB buffer containing 0.5% NaCl, and collect the protein peak. The protein solution is rh-CRT-1. SDS-PAGE electrophoresis is as follows: Figure 3 As shown.

[0091] 1.3rh-CRT-2 Cloning and Expression

[0092] To express the protein in the prokaryotic system, the gene sequence of SEQ ID NO:2 was codon-optimized, and the gene sequence is shown in SEQ ID NO:5. The gene was then subcloned (between BamHI and XhoI) into the pET28a plasmid (a product of Novagen) through whole-gene synthesis, and the plasmid was named pET28a-MCRT.

[0093] Using pET28a-MCRT as a template and pFcrt-2 / pRcrt-2 as upstream and downstream primers, the gene was amplified by PCR, with an NdeI restriction site introduced upstream and an XhoI restriction site introduced downstream.

[0094] pFcrt-2 primer 5'-GTTCATATGGAACCGGCAGTGTATTTTAAG-3' (SEQ ID NO: 6)

[0095] pRcrt-2 primer 5'-GTGCTCGAGACGCTGTTCTTCATCCTGTTTATC-3' (SEQ ID NO:7)

[0096] The CRT gene sequence corresponding to rh-CRT-2 is shown in SEQ ID NO:8, and the protein sequence is shown in SEQ ID NO:9.

[0097] PCR was performed using Thermo Scientific's high-fidelity DNA polymerase Phusion at 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, for 30 cycles, followed by a 10-minute incubation at 72°C. The resulting PCR product (approximately 1060 bp) was purified, double-digested with NdeI / XhoI, and inserted into the plasmid pET42a (Novagen). This plasmid was then transformed into competent *E. coli* TOP10 cells, and positive clones were selected from LB culture containing kanamycin. After successful sequencing, the plasmid was extracted and named pET42a-rh-CRT-2. The plasmid map is shown below. Figure 4 As shown, the plasmid restriction enzyme diagram is as follows: Figure 5As shown. pET42a-rh-CRT-2 transformed into competent BL21(DE3) cells (Thermo Scientific). Positive clones were selected on LB agar plates containing kanamycin sulfate (50 μg / ml). BL21(DE3) positive clones obtained from pET42a-rh-CRT-2 transformation were inoculated into 6.0 ml of LB liquid medium containing 50 μg / ml kanamycin sulfate and incubated at 37°C with shaking at 220 RPM for approximately 3 hours. 2.0 ml of the bacterial culture was then inoculated into a 1.0 L Erlenmeyer flask containing 200 ml of sterile LB medium with a final concentration of 50 μg / ml kanamycin sulfate. Continue incubation at 37℃ and 220 RPM with shaking for 3 hours until the OD600 is approximately 1.0. Then, inoculate 1.0% of the culture into six 5L sterile Erlenmeyer flasks, each containing 1.0L of LB medium. Continue incubation at 30℃ and 220 RPM with shaking. When the OD600 is approximately 0.8, add 1.0M IPTG (AMRESCO product) to bring the final concentration to 0.5mM. Adjust the temperature to 28℃ and continue incubation for 6 hours. Then, centrifuge to collect the bacterial cells.

[0098] 1.4rh-CRT-2 protein purification

[0099] Take 10g of the bacterial cells collected in step 1.3, add 20mM Tris-HCl buffer (pH 7.5) at a ratio of 1:20 (g / g), and sonicate in an ice-water bath. Centrifuge to collect the supernatant. Load the supernatant onto an AKTA purifier 100 system using a GE XK16 / 20 column and GE Ni Sepharose 6FF 10ml packing material. Wash with 20mM Tris-HCl buffer (pH 7.5) containing 100mM imidazole, elute with 25mM EDTA solution, and collect the protein peak. Concentrate the protein peak using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5kDa, load it onto an AKTA purifier 100 system using a GE XK16 / 100 column and GE Sephactyl S-200 packing material. Pack the column to a height of 95cm, elute with 10mM PB buffer (pH 7.2) containing 0.5% NaCl, and collect the protein peak. The protein solution is rh-CRT-2, and SDS-PAGE electrophoresis is as follows: Figure 6 As shown.

[0100] Cloning and expression of 1.5rh-CRT-3

[0101] Using pET28a-MCRT as a template and pFcrt-2 / pRcrt-3 as upstream and downstream primers, the gene was amplified by PCR, with an NdeI restriction site introduced upstream and an XhoI restriction site introduced downstream.

[0102] pFcrt-2 primer 5'-GTTCATATGGAACCGGCAGTGTATTTTAAG-3' (SEQ ID NO: 6)

[0103] pRcrt-3) primer 5'-GTGCTCGAGGTATGCGTAAATAGACGGGTC-3' (SEQ ID NO:10)

[0104] The CRT gene sequence corresponding to rh-CRT-3 is shown in SEQ ID NO:11, and the protein sequence is shown in SEQ ID NO:12.

[0105] PCR was performed using Thermo Scientific's high-fidelity DNA polymerase Phusion at 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, for 30 cycles, followed by a 10-minute incubation at 72°C. The resulting PCR product (approximately 890 bp) was purified, digested with NdeI / XhoI, and inserted into plasmid pET42a. This was then transformed into competent *E. coli* TOP10 cells, and positive clones were selected from LB culture containing kanamycin. After successful sequencing, the plasmid was extracted and named pET42a-rh-CRT-3. The plasmid map is shown below. Figure 7 As shown, the plasmid restriction enzyme diagram is as follows: Figure 8 As shown. pET42a-rh-CRT-3 was used to transform competent BL21(DE3). Positive clones were selected on LB agar plates containing kanamycin (50 μg / ml). BL21(DE3) positive clones obtained from pET42a-rh-CRT-3 transformation were inoculated into 6.0 ml of LB liquid medium containing 50 μg / ml kanamycin sulfate and cultured at 37°C with shaking at 220 RPM for approximately 3 hours. 2.0 ml of the bacterial culture was then inoculated into a 1.0 L Erlenmeyer flask containing 200 ml of sterile LB medium with a final concentration of 50 μg / ml kanamycin sulfate. Continue incubation at 37°C and 220 RPM with shaking for 3 hours until the OD600 is approximately 1.0. Then, inoculate 1.0% of the culture into six 5L sterile Erlenmeyer flasks, each containing 1.0L of LB medium. Continue incubation at 30°C and 220 RPM with shaking. When the OD600 is approximately 0.8, add 1.0M IPTG to bring the final concentration to 0.5mM. Adjust the temperature to 28°C and continue incubation for 6 hours. Then, centrifuge to collect the bacterial cells.

[0106] 1.6rh-CRT-3 protein purification

[0107] Take 10 g of the bacterial cells collected in 1.5, add 20 mM Tris-HCl buffer at pH 7.5 at a ratio of 1:20 (g / g), perform ultrasonic bacterial disruption in an ice-water bath, and collect the supernatant by centrifugation. Load the supernatant onto the AKTA purifier 100 system, select the XK16 / 20 chromatography column from GE Healthcare as the chromatography column, and use 10 ml of Ni sepharose 6FF from GE Healthcare as the packing material. Wash with 20 mM Tris-HCl buffer at pH 7.5 containing 100 mM imidazole, elute with 25 mM EDTA solution, and collect the protein peak. Concentrate the protein peak using an ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa, load it onto the AKTA purifier 100 system, select the XK16 / 100 chromatography column from GE Healthcare, use Sephacryl S-200 from GE Healthcare as the packing material, fill the chromatography column to a height of 95 cm, elute with 10 mM PB buffer at pH 7.2 containing 0.5% NaCl, and collect the protein peak. The protein solution is rh-CRT-3, and the SDS-PAGE electrophoresis is as shown in Figure 9 shown in the figure.

[0108] Example 2

[0109] 1. Experimental study on mouse hair growth

[0110] 1.1 The first experiment

[0111] Experimental animals: Male C57BL / 6 mice, 6 to 7 weeks old, weighing (20±2) g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal production license number SCXK (Beijing) 2021-0006. The animals are housed in a 12 h light / 12 h dark environment with free access to food and water. The mice are divided into a normal control group (normal NC group, NC), a model group (Model), a positive control - minoxidil group (Minoxidil), a positive control - finasteride group (Finasteride), experimental group A and experimental group B. The proteins are stored in a -70℃ refrigerator and diluted with normal saline before use. Experimental group A (rh-CRT-1, the protein concentration is 0.21 mg / ml when in use), experimental group B (rh-CRT-2, the protein concentration is 0.21 mg / ml when in use), with 6 mice in each group.

[0112] The main reagents are as follows: 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., China); isoflurane (RWD Life Science Co., Ltd., China); testosterone (Shanghai Standard Technology Inc., China); paraffin microtome (Thermo Fisher Scientific, USA); small animal anesthesia machine (RWD Life Science Co., Ltd., China); inverted microscope (Olympus Corporation, Japan).

[0113] Modeling and medication: First, a male pattern baldness model was established. One day before modeling, a portion of the hair on the central back of the experimental animals was shaved with a shaving razor, and depilatory cream was applied to the shaved area. 24 hours after depilation, except for the NC group, the other groups received a subcutaneous injection of the modeling agent (testosterone, 5.0 mg / mL) into the neck of mice, with a total dose of 0.2 mL per mouse. Mice in the NC group received an equal volume of physiological saline. After modeling, medication was administered. In the NC group, 0.15 mL of physiological saline was injected once daily. In experimental groups A, B, and the positive control group, the modeling agent (testosterone) was injected 2 hours after each injection, with a dosage of 0.2 mL per animal. In experimental groups A and B, 0.15 mL of the modeling agent was injected into the back once daily. In the positive control minoxidil group, 0.01 mL of 5% minoxidil was applied topically once daily. In the positive control finasteride group, 0.2 mL of finasteride at a concentration of 13 μg / mL was injected per animal once daily.

[0114] result:

[0115] The experiment lasted 21 days, and changes in hair in each group were observed using a dermoscopy. The results are as follows: Figures 10 to 15 As shown: the normal control group had smooth hair; the model group had frizzy hair; compared with the model group, the hair in experimental groups A and B was smoother, and the positive controls—the minoxidil group and the finasteride group—had smooth hair.

[0116] Changes in the hair shaft and cuticle were observed using scanning electron microscopy, and the results are as follows: Figures 16 to 21 As shown: In the normal control group, the hair shaft was covered with layers of overlapping scale-like cuticles, while some hair shafts in the Model group were hollow; the cuticles in both Experimental Group A and Experimental Group B were arranged relatively neatly, with the cuticles in Experimental Group B being denser than those in Group A; the cuticles in the positive control group—minoxidil group—were arranged relatively neatly in a scale-like manner, as were the cuticles in the positive control group—finasteride group.

[0117] 1.2 Second Experiment

[0118] Experimental animals: C57BL / 6 male mice, 6-7 weeks old, weighing (20±2)g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal production license number SCXK (Beijing) 2021-0006. The animals were housed in a 12h light / 12h dark environment, with free access to food and water. The mice were divided into normal control group (normal group, NC); model group (Model); experimental group C (rh-CRT-2, the protein concentration is 0.35 mg / ml when in use), which is set with an injection subgroup and an external smearing subgroup; experimental group D (rh-CRT-3, the protein concentration is 0.21 mg / ml when in use), which is set with an injection subgroup and a smearing subgroup. The proteins for the experimental groups are stored in a -70℃ refrigerator and diluted with normal saline when in use; positive drug control — minoxidil group (Minoxidil). There are 6 mice in each group.

[0119] Model establishment and administration: The alopecia model was established first. One day before the start of model establishment, part of the hair in the central area of the back of the experimental animals was shaved with a surgical blade, and the shaved area was depilated with depilatory cream. 24 hours after depilation, except that the normal control group was given 75% ethanol, the other groups were injected with testosterone solution (5 mg / mL) prepared with 75% ethanol at a dose of 0.01 mL per mouse. 2 hours later, the injection subgroups of experimental group C and experimental group D were injected on the back respectively at a dose of 0.15 mL per mouse, once a day, for 17 consecutive days; the external application subgroups of experimental group C and experimental group D were smeared on the back respectively at a dose of 0.15 mL per mouse, once a day, for 17 consecutive days. The normal control group and the model group were externally applied with normal saline, once a day, 0.15 mL per mouse each time, for 17 consecutive days. During the experimental administration process, except for the normal control group, all other groups were administered 2 hours after the daily injection of the modeling agent (testosterone). Hair conditions were observed with a dermoscope, a color tester, and a scanning electron microscope. Pathological sections were made of the mouse skin after the experiment. The operation of pathological section: The skin pathological tissue was taken and fixed in 4% paraformaldehyde, and the tissue was embedded after trimming, dehydration and clearing. A paraffin microtome was used to cut sections with a thickness of 6 μm, followed by further dewaxing, staining, dehydration, clearing and mounting. Hair follicles in different regions were observed under an optical microscope, and the number of hair follicles was statistically analyzed.

[0120] Scanning electron microscope observation: At the end of the experiment, hair shafts were randomly plucked from the new hair growth area on the back of mice in each group, 3 mice were selected from each group, the plucked hair shafts were fixed, and the changes of hair shafts and hair cuticles were observed through a scanning electron microscope.

[0121] Hormone levels in serum and skin tissue: At the end of the experiment, blood was collected from the orbital region of mice, allowed to stand at room temperature for 30 min, centrifuged at 5000 r / min for 10 min, and the serum was removed. Skin tissue was collected at the end of the experiment and stored at -80℃. Skin tissue was homogenized before the experiment, and changes in serum and skin tissue levels of testosterone, dihydrotestosterone, and estradiol in each group were detected by enzyme-linked immunosorbent assay (ELISA).

[0122] Statistical analysis: All data were processed using SPSS 17.0 software. One-way ANOVA was used for comparisons among multiple groups. Data were expressed as mean ± standard deviation. x (±s) indicates that P<0.05 is statistically significant, while P>0.05 is not considered statistically significant.

[0123] result:

[0124] Dermoscopy was used to observe the entire back skin of mice, and photographs were taken on days 3, 7, 10, 14, and 17 after drug administration. The results are as follows: Figure 22 As shown. On day 17 of the experiment, hair grew on the back skin of the normal control group, while only a small part of the skin in the model group grew hair. Both experimental groups C and D could promote hair growth on the back of mice. Among them, the topical application group of experimental group C had the most significant effect on hair growth, followed by the injection group of experimental group D, the topical application group of experimental group D, and the injection group of experimental group C.

[0125] At the end of the experiment, after shaving and hair removal on the backs of each group, changes in the growth stage and quantity of back hair were observed using a dermoscopy. It was found that the normal control group had abundant and coarse hair, while the model group had sparse, fine, and miniaturized hair. Compared to the model group, the topical application group (Experimental Group C) had increased hair quantity and coarsening, followed by the injection group (Experimental Group D) and the topical application group. The injection group (Experimental Group C) had less hair and finer hair. Figure 23 As shown.

[0126] The changes in the degree of darkening of the back skin in each group were observed using a colorimeter at 7, 10, 14, and 17 days. The results are shown below. Figure 24 :

[0127] 7 days: There was no significant change in the degree of darkening of the back skin in each group (P>0.05).

[0128] 10 days: Compared with the normal control group, the darkness of the back skin in the model group was significantly reduced (P<0.01). Compared with the model group, there was no significant change in the darkness of the back skin in the drug group (P>0.05), while the darkness in the minoxidil group was significantly increased (P<0.01).

[0129] 14 days: Compared with the normal control group, the blackness of the back skin in the model group was significantly reduced (P<0.01), while the blackness in the experimental group C (topical application group) was significantly increased (P<0.05).

[0130] 17 days: The degree of blackening on the back of the model group was significantly lower than that of the normal control group (P < 0.01). Compared with the model group, the degree of blackening in the topical application group (Experimental C) and the injection group (Experimental D) was significantly higher (P < 0.01).

[0131] The changes in hair in each group were observed using scanning electron microscopy, and the results are as follows: Figure 25 As shown, the normal control group had large hair shafts covered with densely packed, overlapping scaly cuticles. The model group had small hair shafts with sparse cuticles and wrinkled surfaces. Compared to the model group, the hair shafts in the topical application group (Experimental C) were significantly thicker, smoother, and had densely packed overlapping scaly cuticles. The hair shafts in the topical application group (Experimental D) were also thicker and had denser cuticles. Although the hair shafts in the injection groups (Experimental C and D) were small, the cuticles were relatively dense. These results indicate that the hair in the topical application group (Experimental C) was significantly better than the model group in both hair shaft diameter and cuticle condition, approaching a healthy state.

[0132] Pathological section results as follows Figure 26 As shown, compared with the normal control group, the number of hair follicles in the model group was significantly reduced, which was statistically significant (P < 0.01). Compared with the model group, both the topical and injection groups in experimental groups C and D significantly promoted the expression of hair follicle numbers in androgenetic alopecia mice, which was statistically significant (P < 0.01), with the topical group in experimental group C showing the most significant increase in hair follicle numbers. These results indicate that both experimental groups C and D can reduce the hair follicle atrophy and reduction in number caused by testosterone, and promote hair growth.

[0133] To observe the effects of the experimental group on different hormones such as testosterone, dihydrotestosterone, and estradiol, blood and skin tissue samples were collected at the end of the experiment to observe changes in serum and skin tissue hormones. The results are as follows: Figure 27 As shown, the results are as follows:

[0134] Compared with the normal control group, serum and skin tissue testosterone levels in the model group were significantly elevated (P < 0.05). Compared with the model group, serum and skin tissue testosterone levels in the injection group (Experimental C group) were significantly lower (P < 0.05), while no statistically significant difference was found in serum and skin tissue levels in other groups (P > 0.05).

[0135] Compared with dihydrotestosterone in the normal control group, dihydrotestosterone in the serum and skin tissues of the model group was significantly increased, with statistical significance (P < 0.01). Compared with the model group, dihydrotestosterone in the serum and skin tissues of the injection subgroup of experimental group C was significantly decreased (P < 0.01), dihydrotestosterone in the serum of the external application subgroup of experimental group C was significantly decreased (P < 0.05), and there was no statistical significance in serum and skin tissues of other groups (P > 0.05).

[0136] Compared with estradiol in the normal control group, there was no change in estradiol in the serum and skin tissues of the model group (P < 0.01). Compared with the model group, there was no statistical significance in estradiol in serum and skin tissues of other groups (P > 0.05), that is, there was no significant change in estradiol among all groups.

[0137] It can be seen from the above results that the external application subgroup of experimental group C can reduce the content of serum dihydrotestosterone, and its effect of promoting hair growth may be related to the reduction of dihydrotestosterone content.

[0138] 1.3 Third experiment

[0139] In the first experiment, the results of experimental group A and experimental group B showed that the hair of the experimental mice was smooth and the hair cuticles were arranged neatly, but from Figure 18 and Figure 19 the comparison, experimental group B (CRT-2, Figure 19 ) had thicker hair shafts than experimental group A (CRT-1, Figure 18 ). Based on the optimization of the mouse AGA (androgenetic alopecia) modeling, this experiment observes the hair growth effects of CRT-2 and CRT-1 on AGA model mice, as well as the effect of the combined application of CRT-2 and the positive control drug minoxidil.

[0140] 1) Experimental animals:

[0141] Male C57BL / 6N mice, 6-7 weeks old, weighing 18-20g, 50 male C57BL / 6N mice, with animal license number SCXK (Beijing) 2021-0006, were housed in a 12h light / 12h dark environment, with free access to food and water. Adaptive feeding was carried out for about 1 week.

[0142] 2) AGA model establishment and administration:

[0143] Using an electric shaver, hair was shaved off in an area of ​​approximately 2cm × 3cm along the spine. An 8% sodium sulfide solution was applied to the shaved area, and after 90±10 seconds, the area was cleaned with a cotton ball soaked in water. Residual sodium sulfide was rinsed off, and the mice were dried. Since the mouse hair follicle cycle was in a quiescent phase at this time, the mouse skin appeared pink. Mice were randomly divided into groups of 10 mice each using Excel software. Modeling was initiated 24 hours later, and the mice were continuously treated for 17 days. An AGA model was established by injecting dihydrotestosterone (DHT) into the experimental mice. 100μL of DHT solution (5mg / kg body weight) was injected subcutaneously into each mouse daily at multiple sites. After 3 hours of absorption, the medication was administered. CRT-2 and CRT-1 protein solutions were applied topically, with 100μL applied. Minoxidil (MNX) was applied topically, with 100μL applied. In the combined CRT and MNX group, CRT-2 protein solution was applied first, followed by MNX two hours later. The solvent formulation was: 50% ethanol, 20% propylene glycol, and 30% purified water. The specific grouping, modeling, and drug administration procedures are shown in Table 1.

[0144] Table 1

[0145]

[0146]

[0147] 3) Hair growth score:

[0148] Scoring is based on the coverage of hair growth:

[0149] 0 points: No hair has grown back, and the skin in the hair removal area is pink;

[0150] 1 point: The hair removal area is gray, and the area coverage E < 20%;

[0151] 2 points: The hair removal area is black, covering 20% ​​of the area. <E<40%);

[0152] 3 points: The hair removal area is black with a few hairs growing, and the area covered is 40%. <E<60%);

[0153] 4 points: The hair removal area is black with some hair growth, and the area covered is 60%. <E<80%);

[0154] 5 points: The hair removal area is black, and the hair coverage area is 80%. <E<100%)。

[0155] Before administering the medication each day, observe the changes in the skin color of the mice. Day 0 is the time when the mice shed their hair, Day 1 is the first day of medication, and so on. Record and analyze the hair regrowth of each mouse on Day 7, Day 9, Day 11, Day 13, Day 15, and Day 17.

[0156] 4) Results:

[0157] On day 17 of drug administration in mice, compared with the model group, all other groups showed significant hair growth, and the differences were statistically significant according to the pre-set scoring values. Results are as follows. Figure 28 As shown in the figure. One-way ANOVA was used to compare the groups. According to the score values, the CRT-2 group was better than the CRT-1 group, and the combination of CRT-2 and MNX was better than the CRT-2 and MNX groups alone, indicating that CRT and MNX have a synergistic effect.

[0158] # Compared with the Model group, the hair growth score was significantly higher in the group using CRT-2 and MNX in combination (P<0.05);

[0159] * Compared with the Model group, the hair growth score of the CRT-2 group was significantly higher (P<0.05);

[0160] ◇Compared with the Model group, the hair growth score of the MNX group was significantly higher (P<0.05);

[0161] ☆Compared with the Model group, the hair growth score of the CRT-1 group was significantly higher (P<0.05); however, there was no significant difference between CRT-1 and MNX (P>0.05);

[0162] ○ Compared with the CRT-1 group, the hair growth score of the CRT-2 group was significantly higher (P<0.05);

[0163] ◇The hair growth score in the CRT-2 and MNX combination therapy group was significantly different from that in the CRT-2 group and the MNX group alone (P<0.05).

[0164] 1.4 Fourth Experiment

[0165] The approach and procedure were basically the same as the third experiment, but the purpose of this experiment was to observe the hair regrowth effect of CRT-3 on AGA (androgenetic alopecia) model mice.

[0166] 1) Laboratory animals:

[0167] The choice of experimental animals was the same as in the third experiment.

[0168] 2) AGA model establishment and drug administration:

[0169] The AGA model was established using the same method as in the third experiment, with the following administration procedure: AGA was established by injecting dihydrotestosterone (DHT) into the mice. Each mouse was given a subcutaneous injection of 100 μL of DHT solution (5 mg / kg body weight) at multiple sites daily, allowing 3 hours for absorption before administration. CRT-3 and CRT-1 protein solutions were applied topically, with 100 μL applied topically, and minoxidil (MNX) was applied topically. The solvent formulation was 50% ethanol, 20% propylene glycol, and 30% purified water. The grouping, modeling, and administration details are shown in Table 2.

[0170] Table 2

[0171]

[0172]

[0173] 3) Hair growth score:

[0174] The scoring criteria and scoring process were the same as in the third experiment.

[0175] 4) Results:

[0176] On day 17 of drug administration in mice, compared with the model group, all other groups showed significant hair growth, and the differences were statistically significant according to the pre-set scoring values. Results are as follows. Figure 29 As shown in the figure. One-way ANOVA was used to compare the groups. According to the score values, the CRT-1, CRT-3 and MNX groups were significantly higher than the Model group; the results of the CRT-3 group were better than those of the CRT-1 group.

[0177] * Compared with the Model group, the hair growth score of the CRT-3 group was significantly higher (P<0.05);

[0178] ◇Compared with the Model group, the hair growth score of the MNX group was significantly higher (P<0.05);

[0179] ☆Compared with the Model group, the hair growth score of the CRT-1 group was significantly higher (P<0.05); however, there was no significant difference between CRT-1 and MNX (P>0.05);

[0180] #Compared with the CRT-1 group, the hair growth score of the CRT-3 group was significantly higher (P<0.05).

[0181] Current researchers generally believe that full-length CRT protein should have better hair growth effects, but the results of the third and fourth experiments show that the truncated CRT fragments CRT-2 and CRT-3 have higher hair growth scores than the full-length CRT protein CRT-1.

[0182] The hair growth score after combining CRT fragment CRT-2 with MNX shows that this combination significantly increases the hair growth score compared to the use of MNX and CRT-2 alone, which fully demonstrates that the combined use of CRT protein and MNX does indeed produce a more superior effect in preventing hair loss and promoting hair growth.

[0183] Glu Lys Gln Met Lys Asp Lys Gln Asp Glu Glu Gln Arg Leu Lys Glu 340 345 350 Glu Glu Glu Asp Lys Lys Arg Lys Glu Glu Glu Glu Ala Glu Asp Lys 355 360 365

[0186] Glu Asp Asp Glu Asp Lys Asp Glu Asp Glu Glu Asp Glu Glu Asp Lys 370 375 380

[0188] Glu Glu Asp Glu Glu Glu Asp Val Pro Gly Gln Ala Lys Asp Glu Leu 385 390 395 400

[0190] <210> 2

[0191] <211> 1200

[0192] <212> DNA

[0193] <213> Artificial Sequence

[0194] <400> 2

[0195] gagcctgccg tctacttcaa ggagcagttt ctggacggag acgggtggac ttcccgctgg 60 atcgaatcca aacacaagtc agattttggc aaattcgttc tcagttccgg caagttctac 120 ggtgacgagg agaaagataa aggtttgcag acaagccagg atgcacgctt ttatgctctg 180 tcggccagtt tcgagccttt cagcaacaaa ggccagacgc tggtggtgca gttcacggtg 240 aaacatgagc agaacatcga ctgtgggggc ggctatgtga agctgtttcc taatagtttg 300 gaccagacag acatgcacgg agactcagaa tacaacatca tgtttggtcc cgacatctgt 360 ggccctggca ccaagaaggt tcatgtcatc ttcaactaca agggcaagaa cgtgctgatc 420 aacaaggaca tccgttgcaa ggatgatgag tttacacacc tgtacacact gattgtgcgg 480 ccagacaaca cctatgaggt gaagattgac aacagccagg tggagtccgg ctccttggaa 540 gacgattggg acttcctgcc acccaagaag ataaaggatc ctgatgcttc aaaaccggaa 600 gactgggatg agcgggccaa gatcgatgat cccacagact ccaagcctga ggactgggac 660 aagcccgagc atatccctga ccctgatgct aagaagcccg aggactggga tgaagagatg 720 gacggagagt gggaaccccc agtgattcag aaccctgagt acaagggtga gtggaagccc 780 cggcagatcg acaacccaga ttacaagggc acttggatcc acccagaaat tgacaacccc 840 gagtattctc ccgatcccag tatctatgcc tatgataact ttggcgtgct gggcctggac 900 ctctggcagg tcaagtctgg caccatcttt gacaacttcc tcatcaccaa cgatgaggca 960 tacgctgagg agtttggcaa cgagacgtgg ggcgtaacaa aggcagcaga gaaacaaatg 1020 aaggacaaac aggacgagga gcagaggctt aaggaggagg aagaagacaa gaaacgcaaa 1080 gaggaggagg aggcagagga caaggaggat gatgaggaca aagatgagga tgaggaggat 1140 gaggaggaca aggaggaaga tgaggaggaa gatgtccccg gccaggccaa ggacgagctg 1200 <210>3

[0196] <211>37

[0197] <212>DNA

[0198] <213>Artificial Sequence

[0199] <400>3

[0200] ctctctcgag aaaagagagc ctgccgtcta cttcaag 37<210>4

[0201] <211>51

[0202] <212>DNA

[0203] <213>Artificial Sequence

[0204] <400>4

[0205] gtaagcggcc gcctaatgat gatgatgatg atgcagctcg tccttggcct g 51 <210>5

[0206] <211>1200

[0207] <212>DNA

[0208] <213>Artificial Sequence

[0209] <400>5

[0210] gaaccggcag tgtattttaa ggagcagttt ttagatggtg atggatggac cagtcggtgg 60 attgagagta agcataagag tgattttgga aaattcgttc tgagtagtgg gaaattttat 120 ggtgatgagg agaaggacaa gggactgcag acgagccagg atgcgcgctt ttatgcactg 180 agcgcatcat tcgaaccgtt tagcaataag ggtcagaccc tggtggtgca gtttacagtt 240 aaacatgagc agaatattga ctgtggtgga gggtatgtta agttatttcc gaatagctta 300 gaccagacgg atatgcatgg tgacagcgaa tacaacatca tgtttggacc cgatatttgt 360 ggtccgggta cgaagaaagt tcatgtgatt tttaattaca agggcaaaaa cgttctgatt 420 aacaaggata tacgttgtaa ggatgatgaa tttacccacc tgtataccct gattgtgcgt 480 cctgataata cctatgaggt taaaattgac aacagtcagg ttgagagcgg ttctctggaa 540 gacgattggg actttctgcc gccgaagaaa atcaaagatc cggacgcaag caaacctgaa 600 gactgggacg agagagcaaa aattgatgat ccgacagata gcaaaccgga agattgggat 660 aaaccggaac atatacctga cccggacgcc aaaaagccag aagattggga cgaagaaatg 720 gatggagaat gggaaccacc ggtgatacaa aatcctgaat acaaaggaga atggaaaccg 780 cgtcagattg acaatccgga ttataaaggc acatggatac atccggaaat agataatccg 840 gaatatagcc cggacccgtc tatttacgca tacgataatt ttggagtgct gggcttagat 900 ttatggcagg tgaaaagcgg cacaattttt gataattttc tgataaccaa cgacgaagca 960 tacgctgaag aatttggtaa cgaaacctgg ggtgtgacca aagcggctga aaaacagatg 1020 aaggataaac aggatgaaga acagcgcctt aaggaggagg aagaagacaa gaaacgcaaa 1080 gaggaggagg aggcagagga caaggaggat gatgaggaca aagatgagga tgaggaggat 1140 gaggaggaca aggaggaaga tgaggaggaa gatgtccccg gccaggccaa ggacgagctg 1200 <210>6

[0211] <211>30

[0212] <212>DNA

[0213] <213>Artificial Sequence

[0214] <400>6

[0215] gttcatatgg aaccggcagt gtattttaag 30<210>7

[0216] <211>33

[0217] <212>DNA

[0218] <213>Artificial Sequence

[0219] <400>7

[0220] gtgctcgaga cgctgttctt catcctgttt atc 33<210>8

[0221] <211>1047

[0222] <212>DNA

[0223] <213>Artificial Sequence

[0224] <400>8

[0225] gaaccggcag tgtattttaa ggagcagttt ttagatggtg atggatggac cagtcggtgg 60 attgagagta agcataagag tgattttgga aaattcgttc tgagtagtgg gaaattttat 120 ggtgatgagg agaaggacaa gggactgcag acgagccagg atgcgcgctt ttatgcactg 180 agcgcatcat tcgaaccgtt tagcaataag ggtcagaccc tggtggtgca gtttacagtt 240 aaacatgagc agaatattga ctgtggtgga gggtatgtta agttatttcc gaatagctta 300 gaccagacgg atatgcatgg tgacagcgaa tacaacatca tgtttggacc cgatatttgt 360 420. ggtccgggta cgaagaaagt tcatgtgatt tttaattaca agggcaaaaa cgttctgatt aacaaggata tacgttgtaa ggatgatga tttacccacc tgtataccct gattgtgcgt cctgataata cctatgaggt taaaattgac aacagtcagg ttgagagcgg ttctctggaa gacgattggg actttctgcc gccgaagaaa atcaaagatc cggacgcaag caaacctgaa gactgggacg agagagcaaa aattgatgat ccgacagata gcaaaccgga agattgggat aaaccggac atatacctga cccggacgcc aaaaagccag aagattggga cgaagaaatg gatggagaat gggaaccacc ggtgatacaa aatcctgaat acaaaggaga atggaaaccg cgtcagattg acaatccgga ttataaaggc acatggatac atccggaaat agataatccg gaatatagcc cggacccgtc tatttacgca tacgataatt ttggagtgct gggcttagat ttatggcagg tgaaaagcgg cacaattttt throwttttc tgataaccaa cgacgaagca 1020. Tacgctgaag aatttggtaa cgaaacctgg ggtgtgacca aagcggctga aaaacagatg aaggataac aggatgaaga acagcgc <210> 9

[0226] <211> 349

[0227] <212>PRT

[0228] <213>Artificial Sequence

[0229] <400>9

[0230] Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly Trp 1 5 10 15 Thr Ser Arg Trp Ile Glu Ser Lys His Lys Ser Asp Phe Gly Lys Phe 20 25 30 Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys Gly 35 40 45

[0234] Leu Gln Thr Ser Gln Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser Phe 50 55 60

[0236] Glu Pro Phe Ser Asn Lys Gly Gln Thr Leu Val Val Gln Phe Thr Val 65 70 75 80

[0238] Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Val Lys Leu Phe 85 90 95 Pro Asn Ser Leu Asp Gln Thr Asp Met His Gly Asp Ser Glu Tyr Asn 100 105 110

[0241] Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val His 115 120 125

[0243] Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp Ile 130 135 140

[0245] Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val Arg 145 150 155 160

[0247] Pro Asp Asn Thr Tyr Glu Val Lys Ile Asp Asn Ser Gln Val Glu Ser 165 170 175 Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys Ile Lys 180 185 190 Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys Ile 195 200 205

[0251] Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu His 210 215 220

[0253] Ile Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu Met 225 230 235 240

[0255] Asp Gly Glu Trp Glu Pro Pro Val Ile Gln Asn Pro Glu Tyr Lys Gly 245 250 255 Glu Trp Lys Pro Arg Gln Ile Asp Asn Pro Asp Tyr Lys Gly Thr Trp 260 265 270

[0258] Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser Ile 275 280 285

[0260] Tyr Ala Tyr Asp Asn Phe Gly Val Leu Gly Leu Asp Leu Trp Gln Val 290 295 300

[0262] Lys Ser Gly Thr Ile Phe Asp Asn Phe Leu Ile Thr Asn Asp Glu Ala 305 310 315 320

[0264] Tyr Ala Glu Glu Phe Gly Asn Glu Thr Trp Gly Val Thr Lys Ala Ala 325 330 335 Glu Lys Gln Met Lys Asp Lys Gln Asp Glu Glu Gln Arg 340 345

[0267] <210>10

[0268] <211>30

[0269] <212>DNA

[0270] <213>Artificial Sequence

[0271] <400>10

[0272] gtgctcgagg tatgcgtaaa tagacgggtc 30 <210>11

[0273] <211>873

[0274] <212>DNA

[0275] <213>Artificial Sequence

[0276] <400>11

[0277] gaaccggcag tgtattttaa ggagcagttt ttagatggtg atggatggac cagtcggtgg 60 attgagagta agcataagag tgattttgga aaattcgttc tgagtagtgg gaaattttat 120 ggtgatgagg agaaggacaa gggactgcag acgagccagg atgcgcgctt ttatgcactg 180 agcgcatcat tcgaaccgtt tagcaataag ggtcagaccc tggtggtgca gtttacagtt 240 aaacatgagc agaatattga ctgtggtgga gggtatgtta agttatttcc gaatagctta 300 gaccagacgg atatgcatgg tgacagcgaa tacaacatca tgtttggacc cgatatttgt 360 ggtccgggta cgaagaaagt tcatgtgatt tttaattaca agggcaaaaa cgttctgatt 420 aacaaggata tacgttgtaa ggatgatgaa tttacccacc tgtataccct gattgtgcgt 480 cctgataata cctatgaggt taaaattgac aacagtcagg ttgagagcgg ttctctggaa 540 gacgattggg actttctgcc gccgaagaaa atcaaagatc cggacgcaag caaacctgaa 600 gactgggacg agagagcaaa aattgatgat ccgacagata gcaaaccgga agattgggat 660 aaaccggaac atatacctga cccggacgcc aaaaagccag aagattggga cgaagaaatg 720 gatggagaat gggaaccacc ggtgatacaa aatcctgaat acaaaggaga atggaaaccg 780 cgtcagattg acaatccgga ttataaaggc acatggatac atccggaaat agataatccg 840 gaatatagcc cggacccgtc tatttacgca tac 873 <210>12

[0278] <211>291

[0279] <212>PRT

[0280] <213>Artificial Sequence

[0281] <400>12

[0282] Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly Trp 1 5 10 15 Thr Ser Arg Trp Ile Glu Ser Lys His Lys Ser Asp Phe Gly Lys Phe 20 25 30 Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys Gly 35 40 45

[0286] Leu Gln Thr Ser Gln Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser Phe 50 55 60

[0288] Glu Pro Phe Ser Asn Lys Gly Gln Thr Leu Val Val Gln Phe Thr Val 65 70 75 80

[0290] Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Val Lys Leu Phe 85 90 95 Pro Asn Ser Leu Asp Gln Thr Asp Met His Gly Asp Ser Glu Tyr Asn 100 105 110

[0293] Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val His 115 120 125

[0295] Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp Ile 130 135 140 Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val Arg 145 150 155 160 Pro Asp Asn Thr Tyr Glu Val Lys Ile Asp Asn Ser Gln Val Glu Ser 165 170 175 Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys Ile Lys 180 185 190 Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys Ile 195 200 205

[0300] Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu His 210 215 220

[0302] Ile Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu Met 225 230 235 240 Asp Gly Glu Trp Glu Pro Pro Val Ile Gln Asn Pro Glu Tyr Lys Gly 245 250 255 Glu Trp Lys Pro Arg Gln Ile Asp Asn Pro Asp Tyr Lys Gly Thr Trp 260 265 270

[0305] Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser Ile 275 280 285

[0307] Tyr Ala Tyr 290

Claims

1. Use of calnexin for the preparation of a product for hair growth, hair development, hair protection or hair loss prevention, characterized in that, The calreticulin is a recombinantly expressed full-length CRT protein, and its amino acid sequence is shown in SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The calreticulin is a protein containing a recombinantly expressed CRT fragment, the amino acid sequence of which is shown in SEQ ID NO:

9.

3. Use according to claim 1, characterized in that, The calreticulin is a protein containing a recombinantly expressed CRT fragment, the amino acid sequence of which is shown in SEQ ID NO:

12.

4. A recombinant protein related to hair growth, hair development, hair protection or hair loss prevention, characterized in that, The recombinant protein is rh-CRT-2 or rh-CRT-3, the amino acid sequence of rh-CRT-2 is shown in SEQ ID NO:9, and the amino acid sequence of rh-CRT-3 is shown in SEQ ID NO:

12.

5. A gene encoding the recombinant protein associated with hair growth, hair development, hair protection and hair loss prevention according to claim 4, characterized in that, The nucleotide sequence encoding rh-CRT-2 is shown in SEQ ID NO:8, and the nucleotide sequence encoding rh-CRT-3 is shown in SEQ ID NO:

11.

6. A plasmid, characterized in that, Genes containing the recombinant proteins of claim 5 related to hair growth, hair development, hair protection, or hair loss prevention.

7. A vector, characterized in that, The gene of the recombinant protein associated with hair growth, hair development, hair protection, or hair loss prevention as described in claim 5 is used as the active ingredient.

8. The use of the recombinant protein of claim 4, the encoding gene of claim 5, the plasmid of claim 6, or the vector of claim 7 in the preparation of pharmaceutical or medical device products for hair growth, hair development, hair protection, or hair loss prevention.

9. Use according to claim 8, wherein the compound is ###0002### The drug is used to improve the condition of the hair shaft and cuticle.

10. The use according to claim 8, wherein the compound is ###00002### The drug described is used to increase the number of hair follicles in the skin.

11. Use according to claim 8, wherein the compound is ###00002### The drug described is one that promotes hair growth by lowering androgen levels.

12. The use according to claim 11, wherein the compound is ###00002### or a pharmaceutically acceptable salt thereof. The male hormone in question is dihydrotestosterone.

13. A product having a hair growth, hair development, hair protection, or hair loss prevention function, characterized by, Its active ingredient is the recombinant protein of claim 4, the gene of claim 5, the plasmid of claim 6, or the vector of claim 7; the use of the product includes at least one of the following: a) Improves hair smoothness; b) Increases hair volume; c) Promotes hair growth; The product is a drug, additive, or active ingredient.

14. A medicament having a hair growth, hair development, hair protection, or hair loss prevention function, characterized by comprising the compound according to claim 1. It consists of the recombinant protein of claim 4, the gene of claim 5, the plasmid of claim 6 or the vector of claim 7, and pharmaceutically additive excipients.

15. The drug with hair growth, hair protection, or hair loss prevention functions according to claim 14, characterized in that, The drug also contains minoxidil, which can be used in combination.

16. The drug with hair growth, hair protection, or hair loss prevention functions according to claim 14, characterized in that, The drug is introduced into the body through injection, spraying, penetration, or absorption.

17. The drug with hair growth, hair protection, or hair loss prevention functions according to claim 14, characterized in that, The drug is introduced into the body tissues through physical or chemical means.

18. The drug with hair growth, hair protection, or hair loss prevention functions according to claim 14, characterized in that, The drug is introduced into the body after being mixed or encapsulated with other substances.

19. The drug with hair growth, hair protection, or hair loss prevention functions according to claim 14, characterized in that, The drug is a topical preparation.

20. The use of calretin in the preparation of medicaments for treating acne or pimples or alopecia areata, characterized in that, The calreticulin CRT is a recombinantly expressed full-length CRT protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

21. The application as described in claim 20, characterized in that, The calreticulin CRT is a protein containing a recombinantly expressed CRT fragment, the amino acid sequence of which is shown in SEQ ID NO:

9.

22. The application as described in claim 20, characterized in that, The calreticulin CRT is a protein containing a recombinantly expressed CRT fragment, the amino acid sequence of which is shown in SEQ ID NO:12.

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