Genetically engineered exosomes loaded with recombinant full-length human collagen type xvii and preparation and use thereof

Abstract

The application provides a kind of genetic engineering exosome loaded with recombinant XVII type full-length human collagen and preparation and application, it is related to the field of bioengineering technology.The genetic engineering exosome provided by the application retains the intracellular domain, transmembrane domain, extracellular domain of XVII collagen completely, and also increases the exosome sorting signal peptide.The application also provides an extraction and separation method of the genetic engineering exosome loaded with recombinant XVII full-length human collagen and application in burn, scald, alopecia, oral ulcer and skin photoaging.The exosome prepared by the application is highly enriched with recombinant XVII type full-length human collagen on the exosome membrane, and retains the physiological activity of XVII type collagen, promotes the proliferation of epidermal cells, fibroblasts and hair follicle stem cells, plays the role of promoting re-epithelialization, promotes wound healing, promotes hair regeneration and delays skin aging.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology engineering, specifically relating to a genetically engineered exosome loaded with recombinant type XVII full-length human collagen, its preparation and application. Background Technology

[0002] Mesenchymal-epithelial interactions (MEAs) play a crucial role in the development of epithelial organs such as the epidermis, hair follicles, and teeth. The thin, sheet-like extracellular matrix basement membrane, located directly beneath the basal surface of epithelial cells, mediates MEAs, allowing epithelial cells to attach to the underlying connective tissue and providing structural support for epithelial repair and regeneration. It also acts as a permeability barrier controlling macromolecular exchange, regulating the passage of substances between the epithelium and supporting tissues, limiting epithelial cell invasion of the underlying tissues, and maintaining the stem cell niche. Hemidesmosomes are subcellular adhesion structures that cover the basement membrane between the mesenchyme and epithelial cells, binding the epithelial cells to the underlying mesenchymal tissue.

[0003] Type XVII collagen is a transmembrane collagen that constitutes hemidesmosomes and plays a crucial role in hemidesmosome stability and epithelial-mesenchymal adhesion. The intracellular domain of type XVII collagen binds to integrin α6β4, bullous pemphigoid antigen, and reticulin, while the extracellular domain binds to laminin-332 and α6 integrin, mediating the adhesion of epidermal keratinocytes and certain other epithelial cells to the underlying basement membrane. Deficiency or loss of function of collagen XVII makes the skin more susceptible to friction damage, leading to the separation of the outermost dermis, such as connective bullous epidermolysis caused by type XVII collagen mutations, manifested as epithelial fragility, easy blistering of the skin and mucous membranes, and enamel hypoplasia. Furthermore, type XVII collagen creates niches for surrounding stem cells, helping to maintain the self-renewal capacity of epidermal cell communities and hair follicle stem cells, and promoting re-epithelialization of damaged skin. A lack of type XVII collagen can cause skin aging, atrophy, fragility, discoloration, hair loss, and slow wound healing, eventually leading to fibrosis and scarring.

[0004] Type XVII collagen is a homotrimer composed of three 180kDa α1 (XVII) chains, containing a globular N-terminal intracellular domain of 466 amino acids, a transmembrane hydrophobic domain of 23 amino acids, and a large rod-shaped C-terminal extracellular domain of 1008 amino acids. The extracellular domain is composed of a triple-helical subcollagenous domain (where X and Y are mostly proline and hydroxyproline residues) assembled from conserved GXY repeat sequences and a non-collagenous domain. The non-collagenous domain is partially N-glycosylated.

[0005] Promoting the expression of type XVII collagen or exogenous supplementation can improve epidermolysis bullosa, promote wound healing, and delay aging and hair loss. For example, by correcting the genes of keratinocytes derived from patients with COL17A1-mutant epidermolysis bullosa and inducing them into pluripotent stem cells, which are then differentiated into keratinocytes expressing type XVII collagen, human skin can be reconstructed in mice; exogenous supplementation with recombinant type XVII collagen can promote wound epithelialization and accelerate wound healing.

[0006] Animal raw material extraction, microbial genetic engineering technology production, and chemically synthesized peptides are the main sources of collagen. However, since type XVII collagen is a large transmembrane protein with glycosylation and a small proportion in animal bodies, and recombinant full-length type XVII collagen is insoluble in water, conventional collagen preparation methods are not suitable for the preparation of biologically active full-length type XVII collagen.

[0007] Exosomes are nanovesicles with a diameter of 40 to 160 nanometers secreted by cells into the extracellular space. They carry various components such as lipids, metabolites, proteins, and RNA, and are an important means of intercellular communication. Exosomes exhibit low immunogenicity and toxicity while possessing high biopermeability and biocompatibility, making them suitable carriers for biomolecule delivery and potential therapeutic pathways. Protein loading onto exosomes occurs on the early endosomal membrane, depending on endosome proteins CD63, Alix, Syntenin-1, and RAB31. Proteins containing amino acid sequences biochemically associated with the KFERQ motif are loaded into exosome subsets. Tagging target proteins with the KFERQ tag can lead to their loading into exosomes, increasing their solubility and enhancing their bioavailability. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a genetically engineered exosome loaded with recombinant type XVII full-length human collagen, along with its preparation and application.

[0009] This application provides a recombinant XVII type full-length human collagen, characterized in that a sorting signal peptide is attached to the N-terminus or C-terminus of the recombinant XVII type full-length human collagen, and the amino acids of the recombinant XVII type full-length human collagen are SEQ ID NO.1 or sequences with more than 75% homology to SEQ ID NO.1.

[0010] SEQ ID NO.1:

[0011] Specifically, the amino acid sequence of the sorting signal peptide is shown in SEQ ID NO.2.

[0012] SEQ ID NO.2: KFERQ.

[0013] In another aspect, the present invention provides a nucleic acid molecule comprising the gene encoding the above-mentioned recombinant type XVII full-length human collagen.

[0014] Specifically, the amino acid sequence of the nucleic acid molecule is shown in SEQ ID NO.3.

[0015] SEQ ID NO.3:

[0016] In another aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecules.

[0017] Specifically, the expression vector is selected from the pEGFP series, pCMV series, pcDNA series, pIRES series, pReceiver series, pLV series, pCDH series, pLenti series, or pFastBac series.

[0018] More specifically, the pEGFP series includes, but is not limited to: pEGFP-C1, pEGFP-N1, pEGFP-B, or pEGFP-T1.

[0019] More specifically, the pCMV series includes, but is not limited to: pCMV-Script, pCMV-Tag1, pCMV-Tag2, or pCMV-Tag3.

[0020] More specifically, the pcDNA series includes, but is not limited to: pcDNA3.1(+), pcDNA3.1(-), pcDNA3.1 / myc-His, pcDNA4 / TO, or pcDNA4 / HisMax.

[0021] More specifically, the pIRES series includes, but is not limited to, pIRES2-EGFP.

[0022] More specifically, the pReceiver series includes, but is not limited to: pReceiver-Lv105, pReceiver-M01, pReceiver-M02, or pReceiver-M03.

[0023] More specifically, the pLV series includes, but is not limited to: pLVX-EF1α-GFP, pLVX-CMV-GFP, or pLVX-Tight-Puro.

[0024] More specifically, the pCDH series includes, but is not limited to: pCDH-CMV-MCS-EF1-Puro, pCDH-EF1-MCS-T2A-copGFP, or pCDH-EF1-MCS-T2A-Puro.

[0025] More specifically, the pLenti series includes, but is not limited to: pLenti-CMV-EGFP or pLenti-CMV-Puro.

[0026] More specifically, the pFastBac series includes, but is not limited to: pFastBac1, pFastBac Dual, or pFastBac HT.

[0027] In another aspect, the present invention provides a host cell comprising the above-mentioned recombinant type XVII full-length human collagen or nucleic acid molecule or expression vector.

[0028] Specifically, the host cell includes mammalian cells, insect cells, or yeast.

[0029] More specifically, the mammalian cells include somatic cells or stem cells.

[0030] Preferably, the stem cells include: induced pluripotent stem cells, umbilical cord blood stem cells, embryonic stem cells, or adult stem cells.

[0031] More preferably, the induced pluripotent stem cells are human induced pluripotent stem cells, mouse induced pluripotent stem cells, dog induced pluripotent stem cells, bovine induced pluripotent stem cells, or porcine induced pluripotent stem cells.

[0032] In some specific embodiments of the present invention, the induced pluripotent stem cells include: induced pluripotent mesenchymal stem cells.

[0033] In another aspect, the present invention provides an engineered exosome, characterized in that the exosome contains the above-mentioned recombinant humanized XVII collagen.

[0034] In another aspect, the present invention provides a method for preparing the above-mentioned engineered exosomes, characterized by comprising the following steps: 1) Construct a vector for expressing recombinant humanized XVII collagen; 2) Transfect the expression vector into host cells; 3) Isolate and collect exosomes from transfected cells.

[0035] On another front, the present invention provides a pharmaceutical composition comprising the above-described recombinant type XVII full-length human collagen or nucleic acid molecule or expression vector or host cell or engineered exosome.

[0036] Specifically, the content of the engineered exosomes in the pharmaceutical composition is 10-1000 μg.

[0037] More specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0038] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0039] More preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0040] In another aspect, the present invention provides the use of the above-mentioned recombinant type XVII full-length human collagen or nucleic acid molecule or expression vector or host cell or engineered exosome or pharmaceutical composition in the preparation of a drug for treating or improving burns, scalds, cuts, hair loss, oral ulcers, bullous skin diseases or photoaging of the skin.

[0041] In another aspect, the present invention provides a treatment method for a disease, the method comprising administering to a subject a therapeutically effective amount of the above-mentioned recombinant type XVII full-length human collagen or nucleic acid molecule or expression vector or host cell or engineered exosome or pharmaceutical composition.

[0042] The diseases mentioned include, but are not limited to: burns, scalds, cuts, hair loss, oral ulcers, bullous skin diseases, or photoaging of the skin.

[0043] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0044] The terms “treat” or “treatment” or “ameliorate” refer to the medical management of a subject’s disease, condition, or undesirable condition. Treatment or preventative benefits include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, condition, or undesirable condition; reduced symptom occurrence; improved quality of life; longer disease-free status; reduction in the severity of the disease, condition, or undesirable condition; stabilization of the disease state; delay in disease progression; remission; survival; prolonged survival; or any combination thereof.

[0045] The term "therapeutic effective dose" refers to a pharmaceutically considered effective dosage, that is, an amount of active drug sufficient to significantly improve the condition without causing serious side effects. Dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the dosage of this invention can vary widely.

[0046] Compared with the prior art, the present invention has the following advantages: The exosomes prepared by this invention are highly enriched with recombinant full-length human type XVII collagen on their membranes and retain the physiological activity of type XVII collagen. This promotes the proliferation of epidermal cells, fibroblasts and hair follicle stem cells, and exerts the effects of promoting re-epithelialization, wound healing, hair regrowth and delaying skin aging. Attached Figure Description

[0047] Figure 1 The results are from the immunoblotting experiment in Example 2.

[0048] Figure 2 The results of electron microscopy experiments on exosomes in Example 3 are shown.

[0049] Figure 3 The results are from the Western blot experiment in Example 3.

[0050] Figure 4 The results of the HaCaT, HDF, HFDPC, and HOK cell proliferation experiments in Example 1 are shown.

[0051] Figure 5 The migration of HaCaT cells in Example 1 after 48 hours is shown.

[0052] Figure 6 The migration of HDF cells in Example 1 after 48 hours is shown.

[0053] Figure 7 The migration of HFDPC cells in Example 1 after 48 hours is shown.

[0054] Figure 8 The relative migration rates of HaCaT, HDF, and HFDPC cells in Effect Example 1 after 48 h are shown. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0056] Example 1: Design of expression vector and construction of plasmid a. The target expression vector consists of: a protein expression sequence, an exosome sorting signal peptide encoding gene (AAGTTTGAAAGACAG), and a recombinant type XVII full-length human collagen encoding gene.

[0057] b. The control expression vector 1 consists of: the gene encoding exosome sorting signal peptide 1 (CCGAGTGCCCCG) and the gene encoding recombinant type XVII full-length human collagen.

[0058] c. The control expression vector 2 consists of: the gene encoding exosome sorting signal peptide 2 (CTGTACCCTCTGCTG) and the gene encoding recombinant type XVII full-length human collagen.

[0059] d. Synthesis of Expression Sequences: The optimized recombinant XVII full-length human collagen sequence, exosome sorting signal peptide 1+ recombinant XVII full-length human collagen sequence, and exosome sorting signal peptide 2 recombinant XVII full-length human collagen sequence were synthesized into their entirety. Homologous recombination cloning was used to clone the recombinant XVII full-length human collagen expression sequence into the pcDNA3.1(+) plasmid, forming the basic vector for the genetically engineered exosome expression plasmid. Primers are shown in Table 1. Table 1

[0060] e. Plasmid Transformation and Extraction: Following standard plasmid transformation procedures, the constructed recombinant plasmid was transformed into DH5α *E. coli* competent cells. The next day, a single colony was picked and inoculated into 3 mL of LB medium, and amplified at 37°C and 200 rpm. After 12 hours, the bacterial culture was inoculated into 200 mL of LB medium and amplified a second time at 37°C and 200 rpm. After 12 hours, plasmid extraction was performed using a plasmid extraction kit (Tiangen Low Endotoxin Medium-Quantity Plasmid Purification Kit) according to the manufacturer's instructions. The concentration of the extracted plasmid was measured, and the sample was filtered through a 0.22 μm filter for sterilization.

[0061] Example 2: Host cell expression of recombinant type XVII full-length human collagen The correctly sequenced plasmid was transfected into 293F cells to detect the expression of recombinant full-length human collagen type XVII. Forty-eight hours after transfection, cells were lysed using pre-chilled RIPA to extract total cellular protein. Western blotting was used to detect the expression of the target protein using an antibody against recombinant full-length human collagen type XVII. The experimental results are shown below. Figure 1 After host cells were transfected with the target expression vector (target sequence), control expression vector 1 (control sequence 1), and control expression vector 2 (control sequence 2), COL17A1 overexpression bands appeared.

[0062] Example 3: Enrichment and Identification of Exosomes Expressing Recombinant Type XVII Full-Length Human Collagen The target expression vector, control expression vector 1, and control expression vector 2 plasmids with correct sequencing were transfected into 293T cells. Four days after transfection, the cell supernatant was collected, and the exosomes in the cell culture supernatant were isolated and purified. The purified exosomes were named target exosome, control exosome 1, and control exosome 2, respectively. The isolation and purification method was PEG precipitation combined with size exclusion chromatography.

[0063] The appearance, particle size, and surface COL17A1 protein of exosomes were characterized using immunoelectron microscopy. 5 μL of the exosome sample was mixed with 5 μL of 4% paraformaldehyde and added to a standard carbon-supported copper mesh (Zhongjing Keyi, 300 mesh BZ11023a). After standing for 15 min, excess sample was aspirated, and 10 μL of 1×PBS containing 0.2% BSA and 5% goat serum was added for blocking. After blocking for 15 min, excess blocking solution was aspirated, and the copper mesh was washed three times with 1×PBS containing 0.1% BSA. 10 μL of COL17A1 polyclonal antibody (Proteintech, 31190-1-AP) (1:100) was added, and the mixture was incubated for 30 min. The copper mesh was then washed three times with 1×PBS containing 0.1% BSA, and Goat anti-Rabbit IgG H&L was added. Conjugated (Bioss, bs-0295G-Gold) (1:200), after incubation for 30 min, the copper mesh was washed three times with 1×PBS containing 0.1% BSA, followed by three washes with deionized water. 2% uranyl acetate was added, and after standing for 20 min, excess liquid was aspirated. The copper mesh was then washed three times with deionized water. The structure of the exosomes and the distribution of recombinant type XVII full-length human collagen on the exosomes were observed under a 120 kV transmission electron microscope (JEM-1400Flash). The experimental results are shown below. Figure 2 .

[0064] like Figure 2 As shown, exosomes derived from 293T cells transfected with control expression vector 1 and control expression vector 2 also expressed COL17A1 protein. However, the exosomes secreted by 293T cells transfected with the target expression vector had denser gold particles on their surface, indicating a higher degree of enrichment of COL17A1 protein (target exosomes) on their surface.

[0065] Immunoblot was used to detect the expression of exosome-specific proteins and recombinant type XVII full-length human collagen. An appropriate amount of the exosome sample was mixed with 5×SDS-PAGE loading buffer and heated in a boiling water bath for 5 min to denature the proteins. An SDS-PAGE gel was prepared and electrophoresis was performed at a constant voltage. Initially, the voltage was adjusted to 80V. When a narrow band formed at the boundary between the separating and stacking gels, the voltage was adjusted to 120V. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel first. A nitrocellulose membrane was prepared and soaked in transfer buffer. The soaked filter paper, nitrocellulose membrane, SDS-PAGE gel, and filter paper were stacked in a bottom-up order, carefully removing air bubbles between each layer. After electrophoresis, the membrane was immersed in 5% skim milk blocking buffer and blocked at room temperature for 2 h. The corresponding antibody was added, and the membrane was incubated overnight at 4℃. The membrane was washed three times with 0.5% PBST for 5 min each time. Add the corresponding HRP-labeled secondary antibody (diluted 1:5000 with washing buffer) and incubate at room temperature with gentle shaking for 1 h. Wash the membrane three times with 0.5% PBST, 5 min each time. Add a 1:1 mixture of chemiluminescent substrates A and B onto the nitrocellulose membrane, gently spreading the luminescent solution evenly with a micropipette to avoid air bubbles. Incubate at room temperature for 5 min, then place the membrane in a chemiluminescence analyzer for imaging. Experimental results are shown in [Figure number missing]. Figure 3 .

[0066] like Figure 3 As shown, exosomes derived from 293T cells transfected with control expression vector 1 and control expression vector 2 also expressed COL17A1 protein, but the exosomes secreted by 293T cells transfected with the target expression vector were enriched with more COL17A1 protein (target sequence EV).

[0067] Comparative Example 1 Compared with the target expression vector in Example 1, the exosome sorting signal peptide encoding gene (AAGTTTGAAAGACAG) remained unchanged, and the amino acid / nucleotide sequence of collagen was replaced with recombinant type III collagen. The constructed plasmid was transfected into H293T cells, and the resulting exosomes were named control exosomes 3.

[0068] Example 1: Efficacy experiment of genetically engineered exosomes We used proliferation and migration experiments of epidermal cells, dermal fibroblasts, dermal papillary hair follicle cells, and oral epithelial cells to verify whether genetically engineered exosomes have the effect of promoting the healing of burns, scalds, and oral ulcers and promoting hair regeneration in vitro. The specific experimental steps are as follows: Cell proliferation experiment: Immortalized keratinocytes (HaCaT cells), dermal fibroblasts (HSF cells), human follicular dermal papillary cells (HFDPC), and oral epithelial cells (HOK) in good growth condition were divided into groups of 3 × 10⁻⁶ cells. 4Seeds were seeded at a density of 1 sample per well in 96-well plates. Complete culture medium was added and the plates were incubated at 5% CO2 and 37°C for 24 hours. After 24 hours, the complete culture medium was removed, and blank (serum-free culture medium), serially diluted test samples (4 sub-wells for each concentration), and control type XVII collagen were added. The plates were incubated at 5% CO2 and 37°C for 24 hours each. CCK8 detection reagent was added according to the instructions, and after 2 hours of incubation, the absorbance at 450 nm was measured using a microplate reader.

[0069] Cell proliferation experiment results as follows Figure 4 As shown, the use of 0.1 mg / mL wild-type 293T cell-derived exosomes (293T EV), COL17A1 (control), and 293T cell-derived exosomes transfected with the target sequence (COL17A1-EV) all promoted the proliferation of epidermal HaCaT cells, dermal fibroblasts (HDF), hair follicle dermal papillary cells (HFDPC), and oral epithelial cells. Among them, the 293T cell-derived exosomes transfected with the target sequence (COL17A1-EV) had a more significant proliferative effect.

[0070] Cell migration assay: Immortalized keratinocytes (HaCaT cells), dermal fibroblasts (HSF cells), and human follicular dermal papillary cells (HFDPC) in good condition were seeded at 80% confluence into 6-well plates. Complete culture medium was added, and the plates were cultured at 5% CO2 and 37°C. When the cells reached 90-100% confluence, scratches were created using a 200 μL pipette tip. After washing away cell debris, blank (serum-free medium), serially diluted test samples (three wells per concentration), and control type XVII collagen were added. The plates were incubated at 5% CO2 and 37°C, and photographs were taken at 0 h and 48 h. Cell migration rate was calculated.

[0071] Cell migration experiment results as follows Figure 5-8 As shown, the use of 0.1 mg / mL of wild-type 293T cell-derived exosomes (293T EV), COL17A1 (control), and 293T cell-derived exosomes transfected with the target sequence (COL17A1-EV) all promoted the migration of epidermal HaCaT cells, dermal fibroblasts (HDF), and hair follicle dermal papillary cells (HFDPC), with the 293T cell-derived exosomes transfected with the target sequence (COL17A1-EV) showing a more significant migration-promoting effect.

[0072] Example 2 Scalding modeling in mice: SD rats weighing 200-250g were selected. 24 hours prior to scalding, their dorsal hair was removed using 8% sodium sulfide. Rats were randomly divided into groups of six: COL17A1-EV treatment group, control exosome 1 treatment group, control exosome 3 treatment group, 293T EV treatment group, COL17A1 treatment group, positive control group for scalding ointment, and negative control group for petroleum jelly. A 2.5cm diameter weight, soaked in 80℃ hot water, was used to scald the mid-posterior dorsal skin of the rats after hair removal. The scalding time was 8 seconds. HE staining and CK19 immunofluorescence staining were performed on the mice. The 8-second scalding group showed complete epidermal damage with some remaining dermis, significant dermal damage below the papillary layer, and very few remaining accessory structures, classifying it as a deep second-degree scald, which was used for further experiments.

[0073] After successful model establishment, medication was administered topically and secured with gauze. Exosomes were dissolved in an appropriate amount of petroleum jelly, with each wound containing 100 μg of exosomes. The positive control group and the petroleum jelly group received the same dosage as the petroleum jelly used to dissolve the exosomes. The medication was changed daily. Specific groupings are shown in Table 2.

[0074] Table 2

[0075] Wound healing photos were taken daily to record wound recovery and hair growth. On days 7, 14, 21, and 28, one mouse was randomly sacrificed to collect skin samples from the burn site. Skin tissue pathological sections were prepared, and the degree of recovery was observed by HE staining of the wound tissue. According to the experimental results, the COL17A1-EV treatment group can significantly promote wound tissue recovery and hair growth.

[0076] Example 3 The therapeutic effects of the exosomes prepared according to this invention on mice with laceration injury were detected according to Table 2. Except for the positive control group which was replaced with rhEGF gel, the other groups remained unchanged. The recovery of the mouse model was observed, including incision healing time, incision tension, collagen deposition, inflammatory infiltration, degree of reepithelialization, expression of PCNA, CD31, and α-SMA.

[0077] The results showed that the exosomes prepared in this invention had a better therapeutic effect on mice with laceration models than other groups.

[0078] Example of effect 4 The therapeutic effects of the exosomes prepared according to this invention on bullous dermatitis model mice were detected according to Table 2. Except for the positive control group, which was replaced with clobetasol propionate cream, the other groups remained unchanged. The improvement of symptoms in bullous dermatitis model mice was observed, including the number of blisters, maximum diameter, total area, time of new blister appearance / rupture, weight change, serum anti-Dsg3 and anti-BP180 IgG titers, skin IgG / C3 deposition intensity (immunofluorescence), peripheral blood IL-6, TNF-α, and IL-17 levels, local neutrophil and eosinophil counts (histology), acantholysis score (percentage of intraepidermal cleft length), subepidermal blister depth and basement membrane integrity, and scratching frequency (pruritus behavior).

[0079] The results showed that the exosomes prepared by this invention could effectively alleviate the symptoms of bullous dermatitis model mice, and the therapeutic effect was better than that of other groups.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A recombinant type XVII full-length human collagen, characterized in that, The recombinant XVII type full-length human collagen is attached to a sorting signal peptide at its N-terminus or C-terminus, and the amino acids of the recombinant XVII type full-length human collagen are SEQ ID NO.1 or sequences with more than 75% homology to SEQ ID NO.

1.

2. The recombinant type XVII full-length human collagen according to claim 1, characterized in that, The amino acid sequence of the sorting signal peptide is shown in SEQ ID NO.

2.

3. A nucleic acid molecule, characterized in that, The nucleic acid encodes the recombinant type XVII full-length human collagen as described in any one of claims 1-2.

4. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule as described in claim 3.

5. The expression vector according to claim 4, characterized in that, The expression vector is selected from the pEGFP series, pCMV series, pcDNA series, pIRES series, pReceiver series, pLV series, pCDH series, pLenti series, or pFastBac series.

6. The expression vector according to claim 5, characterized in that, The pcDNA series includes: pcDNA3.1(+), pcDNA3.1(-), pcDNA3.1 / myc-His, pcDNA4 / TO, or pcDNA4 / HisMax.

7. A host cell, characterized in that, The host cell contains the recombinant full-length human collagen of type XVII as described in any one of claims 1-2, or the nucleic acid molecule as described in claim 3, or the expression vector as described in any one of claims 4-6.

8. The host cell according to claim 7, characterized in that, The host cell is a eukaryotic host cell.

9. The host cell according to claim 8, characterized in that, The host cells include mammalian cells, insect cells, or yeast.

10. The host cell according to claim 9, characterized in that, The mammalian cells include somatic cells or stem cells.

11. The host cell according to claim 10, characterized in that, The stem cells include: induced pluripotent stem cells, umbilical cord blood stem cells, embryonic stem cells, or adult stem cells.

12. The host cell according to claim 11, characterized in that, The induced pluripotent stem cells are human induced pluripotent stem cells, mouse induced pluripotent stem cells, dog induced pluripotent stem cells, bovine induced pluripotent stem cells, or porcine induced pluripotent stem cells.

13. The host cell according to claim 12, characterized in that, The induced pluripotent stem cells include: induced pluripotent mesenchymal stem cells.

14. An engineered exosome, characterized in that, The exosomes comprise the recombinant humanized XVII collagen as described in any one of claims 1-2.

15. The method for preparing engineered exosomes according to claim 14, characterized in that, Includes the following steps: 1) Construct a vector for expressing recombinant humanized XVII collagen; 2) Transfect the expression vector into host cells; 3) Isolate and collect exosomes from transfected cells.

16. A pharmaceutical composition, characterized in that, Includes the recombinant type XVII full-length human collagen as described in any one of claims 1-2, or the nucleic acid molecule as described in claim 3, or the expression vector as described in any one of claims 4-6, or the host cell as described in any one of claims 7-13, or the engineered exosome as described in claim 14.

17. The pharmaceutical composition according to claim 16, characterized in that, The content of the engineered exosomes in the pharmaceutical composition is 10-1000 μg.

18. The pharmaceutical composition according to claim 17, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

19. The use of the recombinant type XVII full-length human collagen of any one of claims 1-2, or the nucleic acid molecule of claim 3, or the expression vector of any one of claims 4-6, or the host cell of any one of claims 7-13, or the engineered exosome of claim 14, or the pharmaceutical composition of any one of claims 16-18 in the preparation of a medicament, said medicament being used to treat or improve burns, scalds, cuts, hair loss, oral ulcers, bullous skin diseases, or photoaging of the skin.

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