CN114794017B

CN114794017B - Construction method of pemphigus vulgaris animal model

Info

Publication number: CN114794017B
Application number: CN202210523319.7A
Authority: CN
Inventors: 陆前进; 高长醒; 刘梅; 张博; 辛月; 王来; 尹昊媛
Original assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Current assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2022-11-22
Anticipated expiration: 2042-05-13
Also published as: CN114794017A

Abstract

The invention discloses a construction method of an ordinary pemphigus animal model, which comprises the steps of utilizing a vaccination technology, taking desmosomucoprotein 3 as a vaccine antigen, and starting to show a phenotype in 28-35 days after primary immunization/boosting immunization of a wild mouse, wherein the phenotype comprises blisters, exfoliation and skin ulceration generated on the hind legs and the tail of the mouse with the integral phenotype; serum autoantibodies; the histological examination shows that the epidermolysis of the epidermis acanthosis, antibody deposition and other pemphigus vulgaris are changed, partial mice show disease phenotypes such as local skin depilation, ulceration, keratoconjunctivitis and the like, the clinical symptoms of the pemphigus vulgaris are simulated, and an important tool is provided for the immunotherapy of the pemphigus vulgaris.

Description

Construction method of pemphigus vulgaris animal model

Technical Field

The invention belongs to the technical field of construction methods of animal models of diseases, and particularly relates to construction of an animal model of pemphigus vulgaris.

Background

Pemphigus (pemphigus) is a group of autoantibody-mediated, serious, life-threatening autoimmune skin diseases, including Pemphigus Vulgaris (PV), pemphigus foliaceus (pemphigus folliculeus), pemphigus paraneoplastic (paraneoplastic pemphigus), etc. [1-3]. In PV patients, autoreactive plasma cells exist, and the secreted autoantibodies are combined with desmoglein3 (Dsg 3), which is a cell surface adhesion protein of the epidermal layer of the skin, so that the acanthosis is loosened, and the symptoms of skin blister, repeated ulceration and the like are formed. The breakdown of the skin barrier not only causes pain and increases the chance of pathogen infection, but also affects the appearance and increases the psychological burden on the patient. The development of an animal model which can simulate clinical symptoms and has cost advantages is of great significance for researching a safe and effective novel treatment method for pemphigus.

An active PV mouse model induced by adoptive transfer is an international common PV adult mouse model, spleen lymphocytes of a Dsg3 knockout mouse are used as a donor, an immunodeficiency mouse is used as a receptor, the receptor mouse can develop phenotypes such as weight loss, hair loss, local skin tissue damage and the like after adoptive transfer for 2-4 weeks, anti-Dsg 3 antibodies can be detected in serum, and skin tissue examination shows that epidermal layer keratinocyte has antibody deposition to cause adhesion loss and acantholysis [4,5]. However, the model needs a gene knockout mouse and an immunodeficient mouse, the cost is high, and the application of the model in the aspects of immunotherapy and vaccine development is limited by the immunodeficiency existing in a receptor mouse.

Active immune induction is an important method for modeling an autoimmune disease animal model, is widely applied to experimental research on autoimmune diseases such as multiple sclerosis and arthritis, and has no report on an active immune induced PV animal model. Masayuki Amagai of Keio University in Japan reported that active immunization of wild-type mice (C57 BL/6N, BALB/C and C3H/HeJ) with recombinant Dsg3 protein could not induce the production of antibodies recognizing Dsg3 in the natural state [6], presumably due to the self-expression of Dsg3 by wild-type mice, which is immune-tolerant to Dsg 3. Considering the earlier reports in this document [6], while the available immune adjuvants are limited, mainly aluminum adjuvant, freund adjuvant, etc., during more than 20 years thereafter, with the gradual and intensive pattern recognition receptor research, a variety of immune adjuvants with strong innate immune cell activation have been approved for clinical use or widely used in experimental research, such as TLR4 agonist monophosphoryl lipid a (MPLA) in cervical cancer vaccine, TLR9 agonist oligonucleotide 1018 (CpG-ODNs 1018) in hepatitis b vaccine, MF59 adjuvant (squalene-based oil-in-water nanoemulsion activates innate immune system) in influenza vaccine, etc. [7]. In combination with the subsequent case report of pemphigus after herpes zoster vaccine inoculation [8], we speculate that over-activation of innate immunity may break through the body's immune tolerance to self-antigens, leading to the development of autoimmune skin diseases. In the invention, a construction method of an active immune induced PV animal model is established so as to solve the difficulty that the current adoptive transfer induced PV model depends on gene knockout mice and immunodeficient mice.

The above references are:

1.Zhao W.,Wang J.,Zhu H.,and Pan M.,Comparison of Guidelines for Management of Pemphigus:a Review of Systemic Corticosteroids,Rituximab,and Other Immunosuppressive Therapies.Clin Rev Allergy Immunol,2021.61(3):p.351-362.PMID:34350539.DOI:10.1007/s12016-021-08882-1.

2.Egami S.,Yamagami J.,and Amagai M.,Autoimmune bullous skin diseases,pemphigus and pemphigoid.J Allergy Clin Immunol,2020.145(4):p.1031-1047.PMID:32272980.DOI:10.1016/j.jaci.2020.02.013.

3.Yang M.,Wu H.,Zhao M.,Chang C.,and Lu Q.,The pathogenesis of bullous skin diseases.J Transl Autoimmun,2019.2:p.100014.PMID:32743502.DOI:10.1016/j.jtauto.2019.100014.

4.Kasperkiewicz M.,Ellebrecht C.T.,Takahashi H.,Yamagami J.,Zillikens D.,Payne A.S.,and Amagai M.,Pemphigus.Nat Rev Dis Primers,2017.3:p.17026.PMID:28492232.DOI:10.1038/nrdp.2017.26.

5.Schmidt E.,Kasperkiewicz M.,and Joly P.,Pemphigus.Lancet,2019.394(10201):p.882-894.PMID:31498102.DOI:10.1016/S0140-6736(19)31778-7.

6.Amagai M.,Tsunoda K.,Suzuki H.,Nishifuji K.,Koyasu S.,and Nishikawa T.,Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus.J Clin Invest,2000.105(5):p.625-31.PMID:10712434.DOI:10.1172/jci8748.

7.Pulendran B.,P S.A.,and O'Hagan D.T.,Emerging concepts in the science of vaccine adjuvants.Nat Rev Drug Discov,2021.20(6):p.454-475.PMID:33824489.DOI:10.1038/s41573-021-00163-y.

8.Bell H.,Kamal N.,and Wong U.,Blistering autoimmune skin reaction following SHINGRIX vaccination in an ulcerative colitis patient:Case report and literature review.Vaccine,2020.38(47):p.7455-7457.PMID:33067034.DOI:10.1016/j.vaccine.2020.09.073.

disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing an animal model of pemphigus vulgaris aiming at the defects of the prior art, and provides a new animal model for the research and development of PV antigen-specific therapy.

In order to achieve the technical purpose, the invention discloses a construction method of an pemphigus vulgaris animal model, and particularly, disease phenogenesis is mediated by inoculating a vaccine to a wild mouse, wherein the vaccine comprises an antigen and an immunologic adjuvant, and the antigen component is desmoglein 3; the immunological adjuvant is selected from any one or combination of several of the following groups: dsRNA analogs, DNA analogs, monophosphoryl lipid a analogs, and squalene.

The wild type mouse is a C57BL/6J mouse, and the week age is 4-8 weeks.

Preferably, the dsRNA analogue is any one of polyinosinic acid cytidylic acid and polyadenylic acid uridylic acid; the DNA analogue is any one of CpG 1018, cpG 1466, cpG 1555 and CpG 1826; the monophosphoryl lipid A analogue is any one of MPLA, RC529 and OM 174; the squalene is MF59 or AS03.

Further preferably, the immunological adjuvant is any one of MPLA, a mixture of Poly (I: C) and CpG1826, addaVax. When the immunoadjuvant is a mixture of Poly (I: C) and CpG1826, preferably the ratio of the two is 5. Poly (I: C) and CpG1826 act on TLR3 and TLR9, respectively, and can synergistically induce stronger antigen presenting cells and higher T cell quality compared with the single use of the antigen presenting cells.

The route of the vaccination is any one of hypodermic injection with or without needle, intradermal injection, intraperitoneal injection, intramuscular injection, lymph node injection, intravenous injection or skin application.

The invention further establishes a construction method of the pemphigus vulgaris animal model, which comprises the following steps:

(1) Mixing the recombinant desmosomal protein 3 with an immunologic adjuvant to prepare a vaccine taking the recombinant desmosomal protein 3 as an antigen;

(2) Inoculating the vaccine prepared in the step (1) to a wild mouse, and inoculating according to a primary immunization mode and a boosting immunization mode;

(3) The mice develop a disease phenotype 28-35 days after the initial immunization.

Wherein, in the step (1), the immunologic adjuvant is MPLA, addaVax or a mixture of poly (I: C) and CpG 1826.

Preferably, when the immunoadjuvant is MPLA, the recombinant protein vaccine is prepared according to the proportion of 3.5-50 μ g of recombinant desmoglein and 0.5-50 μ g of MPLA; when the immunologic adjuvant is a mixture of poly (I: C) and CpG1826, the recombinant protein vaccine is prepared according to the proportion of 3.5-50 mug of recombinant desmosomal mucin, 3-300 mug of poly (I: C) and 0.1-10 mug of CpG 1826; when the immunological adjuvant is AddaVax, the recombinant protein vaccine is configured according to the volume ratio of the recombinant desmoglein3 to AddaVax of 1.

In the step (2), after the wild type mice are anesthetized in the primary immunization, the vaccines are continuously inoculated for 3 days, and after the 14 th day of inoculation, the vaccines are continuously inoculated for 3 days again in the boosting immunization.

Preferably, the inoculation mode in the step (2) is subcutaneous inoculation.

Preferably, the dose for each injection is 3.5-50 μ g recombinant desmoglein, 0.5-50 μ g MPLA, 3-300 μ g poly (I: C), 0.1-10 μ g CpG1826, 25 μ l AddaVax.

Has the advantages that: the invention constructs the pemphigus vulgaris animal model by selecting proper adjuvant and vaccine antigen, does not need gene knock-out mice and immunodeficient mice, can specifically induce skin tissues to generate PV symptoms, and provides an important tool for immunotherapy of pemphigus vulgaris.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a graph of mouse-induced pemphigus vulgaris disease phenotype 35 days after vaccination with recombinant Dsg3 protein vaccine adjuvanted with monophosphoryl lipid A (MPLA), wherein A is the overall appearance of the mouse and arrows indicate the site of skin ulceration or epidermal deletion; panel B shows ocular keratoconjunctivitis in some mice; panel C shows the visible exfoliation, ulceration of the tail skin, and antibody deposition between epidermal keratinocytes and loosening of the stratum corneum as indicated by histological examination; panel D shows the apparent exfoliation of the skin, ulceration of the skin, and the deposition of antibodies between epidermal keratinocytes and the loosening of the acanthosis epidermis, as indicated by histological examination, in the feet of the mice; the graph E shows the partial visible skin lesions, hair loss, and the histological examination of the mice with the scale of 100 microns showing the antibody deposition between epidermal keratinocytes and the loosening of epidermal spinous layer; panel F shows the level of anti-Dsg 3 antibodies in the mouse serum measured by ELISA;

FIG. 2 shows mouse-induced pemphigus vulgaris-like disease phenotype 35 days after vaccination with recombinant Dsg3 protein vaccine using Polyinosinic-polycytidic acid, poly (I: C) and oligonucleotide 1826 (cytotoxic phosphoguanosine-conjugating oligonucleotide, cpG-ODNs 1826) as immune adjuvant, panel A shows mouse overall appearance phenotype, arrows show skin ulceration or epidermal deletion; panel B shows ocular keratoconjunctivitis in some mice; the figure C shows that the tail of the mouse is visually provided with the exfoliation of the epidermis, the skin ulceration and the antibody deposition and the acanthosis of the epidermis between the keratinocytes as shown by the histological examination; d is the epidermal exfoliation, skin ulceration, visible on the foot of mice, and antibody deposition and release of the epidermal acanthosis between epidermal keratinocytes, indicated by histological examination, on a scale of 100 μm; e is a graph showing the local appearance of skin lesions, hair loss in mice; panel F shows the level of anti-Dsg 3 antibodies in the sera of mice measured by ELISA.

FIG. 3 is a 35 day post-vaccination with recombinant Dsg3 protein vaccine adjuvanted with AddaVax, showing the mouse's overall appearance phenotype, arrows indicate skin ulceration or epidermal deletion sites; b is the mouse tail part, the pelma appearance can be seen, the epidermis is fallen, the skin is ulcerated; panel C shows the ELISA to measure anti-Dsg 3 antibody levels in mouse serum.

FIG. 4 is the overall appearance phenotype of mice 35 days after vaccination with recombinant Dsg3 protein vaccine adjuvanted with aluminum adjuvant (Alum), freund's adjuvant (CFA). The A diagram shows the overall appearance phenotype of the mouse, and the B diagram shows the appearance phenotype of the tail and the sole of the mouse.

Detailed Description

The present invention will be further described with reference to the following examples, but it should be understood that the scope of the present invention as defined above is not limited to the following examples. Various alterations and modifications can be made without departing from the technical spirit of the invention, and the scope of the invention is intended to be covered by the invention in accordance with the general technical knowledge and common practice in the field.

Example 1 MPLA is an immunoadjuvant recombinant protein vaccine induced PV model.

1. Experimental Material

SPF grade 7 week female C57BL/6J mice, recombinant Dsg3 protein solution (Nanjing Minn.), MPLA (invivogen), 4% paraformaldehyde (Biosharp), OCT embedding medium (Sakura), goat anti-mouse IgG-FITC, goat anti-mouse IgG-HRP (Abclonal), and the like.

2. Experimental methods

1. Mouse vaccination

And (4) preparing the recombinant protein vaccine. After anesthesia, the mice were inoculated with 50. Mu.l of recombinant protein vaccine (containing 5. Mu.g each of Dsg3 protein and MPLA) subcutaneously on the sole of the foot for 3 days continuously. On day 14, the mice were boosted and inoculated for 3 days in the same manner.

2. Extraction and histological detection of skin tissues of rat tail, sole and back

On day 35, mice were sacrificed by dislocation of the cervical vertebrae after anesthesia. Taking off skin of rat tail, foot sole and back with sharp scalpel and ophthalmic scissors about 0.5 cm × 0.5 cm, spreading on tin foil paper, and quick freezing with liquid nitrogen or fixing in 4% paraformaldehyde.

The skin tissue after the liquid nitrogen quick freezing is embedded by OCT, and then placed on a freezing section mould, after being sliced by a freezing microtome, the OCT is washed off, goat anti-mouse IgG-FITC (1) is added, and the mixture is incubated for 35 minutes in a dark place at 37 ℃, washed for 2 times and then imaged by a fluorescence microscope.

Fixing the extracted skin tissue with 4% paraformaldehyde, dehydrating, soaking in wax, and embedding. After the tissue blocks are sliced, the tissue blocks are spread, baked, dewaxed, stained with hematoxylin and eosin, and photographed in a selected visual field by a microscope after being sealed.

3. Enzyme-linked immunosorbent assay

Sera were prepared at different time points, split and frozen for ELISA. Prepare 50. Mu.g/ml of Dsg3 solution, add 100. Mu.l/well to the plate, coat overnight at 4 ℃, wash 3 times the next day with wash solution, incubate 2 hours at room temperature with 200. Mu.l blocking solution (1% BSA in PBS), wash 3 times, and pat dry for use. The diluted serum to be tested was added at 50-100. Mu.l/well and incubated at room temperature for 2 hours. Washed 3 times, patted dry, goat anti-mouse IgG-HRP (1. Washing for 5-7 times, drying, adding TMB color developing solution, developing at room temperature for 30 min, and detecting OD value at 450nm with enzyme labeling instrument.

3. Results of the experiment

We found that pemphigus-like changes such as blisters, excoriations, skin ulcerations and the like can be induced in hind paw and tail of wild type C57BL/6J mice after plantar subcutaneous injection, priming and boosting inoculation by using TLR4 agonist MPLA as an immunoadjuvant and rDsg3 as an antigen (FIG. 1. A), and symptoms such as local skin depilation, ulceration, keratoconjunctivitis and the like appear in some mice (FIG. 1.B, E). The direct immunofluorescence result shows that the epidermal cells of the skin tissue have positive staining, which indicates that the epidermal layer has antibody deposition; HE staining results also suggested that active immunization induced pemphigus pathology typical of acantholysis (fig. 1.C, d, e). ELISA results showed that the serum Dsg 3-specific antibody of the model mouse was significantly higher than that of the untreated mouse, suggesting that the combination of rSg 3 and immunoadjuvant can induce the production of autoantibody (FIG. 1. F). The above results indicate that the animal model phenotype is similar to the clinical symptoms of PV.

Example 2 Poly (I: C) and CpG1826 as immunoadjuvants of recombinant protein vaccine induced PV model.

1. Experimental Material

SPF grade 7 week female C57BL/6J mice, recombinant Dsg3 protein solution (Nanjing Minn research), poly (I: C) (invivogen), cpG1826 (Biotech), 4% paraformaldehyde (Biosharp), OCT embedding medium (Sakura), goat anti-mouse IgG-FITC, goat anti-mouse IgG-HRP (Abclonal), and the like.

2. Experimental method

1. Mouse vaccination

The recombinant protein vaccine is prepared, and after the mice are anesthetized, 50 mu l of the recombinant protein vaccine (containing 5 mu g of Dsg3 protein, 30 mu g of poly (I: C) and 1821 mu g of CpG) is inoculated subcutaneously on the soles of the feet, and the inoculation is continuously carried out for 3 days. On day 14, the mice were boosted and inoculated for 3 days in the same manner.

On day 35, mice were sacrificed by dislocation of the cervical vertebrae after anesthesia. Taking off skin of rat tail, foot sole and back with sharp scalpel and ophthalmologic scissors, spreading on tin foil paper at a thickness of about 0.5 cm × 0.5 cm, and quick freezing with liquid nitrogen or fixing in 4% paraformaldehyde.

The skin tissue after the liquid nitrogen quick freezing is embedded by OCT, then placed on a freezing section mould, sliced by a freezing microtome, washed off the OCT, added with goat anti-mouse IgG-FITC (1 100), incubated for 35 minutes at 37 ℃ in a dark place, washed for 2 times and imaged by a fluorescence microscope.

3. Enzyme-linked immunosorbent assay

Sera were prepared at different time points, split and frozen for ELISA. Prepare 50. Mu.g/ml of Dsg3 solution, add 100. Mu.l/well to the plate, coat overnight at 4 ℃, wash 3 times the next day with wash solution, incubate 2 hours at room temperature with 200. Mu.l blocking solution (1% BSA in PBS), wash 3 times, and pat dry for use. The diluted serum to be tested is added into the mixture according to the concentration of 50-100 mu l/hole, and the mixture is incubated for 2 hours at room temperature. Washed 3 times, patted dry, added goat anti-mouse IgG-HRP (1. Washing for 5-7 times, drying, adding TMB developing solution, developing at room temperature for 30 min, and detecting OD value at 450nm with enzyme-labeling instrument.

3. Results of the experiment

We found that combination of TLR3 agonist Poly (I: C) and TLR9 agonist CpG1826 as immunological adjuvant and rDsg3 as antigen, via plantar subcutaneous injection and priming/boosting inoculation, it can induce pemphigus-like changes such as blister, epidermolysis, skin ulceration bleeding and the like on hind paw and tail of wild type C57BL/6J mice (figure 2. A), and some mice have symptoms such as local skin depilation, ulceration, keratoconjunctivitis and the like (figure 2.B, E). The direct immunofluorescence result shows that the epidermal cells of the skin tissue have positive staining, which indicates that the epidermal layer has antibody deposition; HE staining results also suggested that active immunization induced pemphigus pathology typical of acantholysis (fig. 2.C, d). ELISA results showed that Dsg 3-specific antibodies were significantly elevated in serum from model mice 21 days and 35 days after vaccination compared to untreated mice, suggesting that combination of rDsg3 and immune adjuvant could induce autoantibody production (fig. 2. F). The above results indicate that the animal model phenotype is similar to the clinical symptoms of PV.

Example 3 AddaVax is a recombinant protein vaccine-induced PV model of an immunoadjuvant.

1. Experimental Material

SPF grade 7 week female C57BL/6J mice, recombinant Dsg3 protein solution (Nanjing Minn.), addaVax adjuvant (invivogen), goat anti-mouse IgG2C-HRP (Southern Biotech), and the like.

2. Experimental methods

1. Mouse vaccination

The recombinant protein vaccine is prepared according to the volume ratio of Dsg3 protein to AddaVax of 1. After the mice were anesthetized, 50. Mu.l of recombinant protein vaccine (containing 5. Mu.g of Dsg3 protein and 25. Mu.l of AddaVax) was subcutaneously inoculated on the sole of the foot for 3 consecutive days. On day 14, the mice were boosted and inoculated for 3 days in the same manner.

2. Enzyme-linked immunosorbent assay

Sera were prepared at different time points, split and frozen for ELISA. Prepare 50. Mu.g/ml of Dsg3 solution, add 100. Mu.l/well to the plate, coat overnight at 4 ℃, wash 3 times the next day with wash solution, incubate 2 hours at room temperature with 200. Mu.l blocking solution (1% BSA in PBS), wash 3 times, and pat dry for use. The diluted serum to be tested was added at 50-100. Mu.l/well and incubated at room temperature for 2 hours. Washed 3 times, patted dry, goat anti-mouse IgG2c-HRP (1. Washing for 5-7 times, drying, adding TMB developing solution, developing at room temperature for 30 min, and detecting OD value at 450nm with enzyme-labeling instrument.

3. Results of the experiment

We found that by using AddaVax as an immunoadjuvant and rDsg3 as an antigen, through plantar subcutaneous injection, priming and boosting, pemphigus-like changes such as blisters, excoriations, skin ulcerations and the like were induced in the hind paw and tail of wild type C57BL/6J mice, whereas mice given rDsg3 alone and AddaVax alone had intact skin and no disease phenotype and no difference in appearance from untreated mice (FIG. 3.A, B). ELISA results showed that Dsg3 specific antibodies in the serum of model mice were significantly higher than untreated mice, mice given rDsg3 only and mice given adavax only 14 days, 42 days after vaccination (fig. 3. C). It is noteworthy that although mice given rDsg3 alone also produced anti-rDsg 3 antibodies, they did not display a disease phenotype, suggesting that in addition to autoantibodies, immune adjuvant-mediated innate immune system or cellular immunity also plays an important role in the development of PV disease. The above results indicate that a recombinant protein vaccine with AddaVax as an immunoadjuvant can induce the PV disease phenotype.

Example 4 aluminum adjuvant and freund's adjuvant failed to induce the PV model.

1. Experimental materials

SPF grade 7 week female C57BL/6J mice, recombinant Dsg3 protein solution (Nanjing Minn.), aluminum adjuvant Alum (invitrogen), freund's complete adjuvant CFA (invitrogen), and the like.

2. Experimental methods

1. Mouse vaccination

The recombinant protein vaccine is prepared according to the proportion that Dsg3 protein and Alum or CFA have the volume of 1. After mice were anesthetized, they were inoculated subcutaneously with 50. Mu.l of recombinant protein vaccine (containing Dsg3 protein 5. Mu.g, alum or CFA 25. Mu.l) for 3 consecutive days. On day 14, the mice were boosted and inoculated for 3 days in the same manner.

2. Mouse phenotype observations

At various time points after inoculation, the integrity of the skin tissue of the hind paw, tail and back of the mice was observed.

3. Results of the experiment

We found that after immunization and booster inoculation with Alum or CFA as the immunoadjuvant and rDsg3 as the antigen by subcutaneous injection on the sole, wild type C57BL/6J mice could not be induced to develop pemphigus-like changes such as blisters, excoriation, ulceration of the skin, and the skin of the mice was intact, which was no different from the appearance of untreated mice, and only swelling of the sole due to inoculation or Alum deposition was observed (FIG. 4.A, B). The above results indicate that Alum and CFA cannot be induced to produce a PV disease phenotype upon vaccination.

The present invention provides a method and a concept for preparing animal models of pemphigus vulgaris by active immunization, and a method and a way for implementing the technical scheme are many, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1.A method of constructing an animal model of pemphigus vulgaris, wherein the phenotyping of a disease is mediated by vaccinating a wild-type mouse, wherein the vaccine comprises an antigen and an immunoadjuvant, and the antigen component is recombinant desmoglein 3; the immunological adjuvant is any one of MPLA, a mixture of Poly (I: C) and CpG1826 and AddaVax.

2. The method for constructing a recombinant human milk according to claim 1, wherein the wild-type mouse is a C57BL/6J mouse, and the week-old mouse is 4-8 weeks.

3. The method according to claim 1, wherein the vaccination is performed by any one of subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular injection, intralymphatic injection, intravenous injection, or skin application with or without needles.

4.A method for constructing an animal model of pemphigus vulgaris is characterized by comprising the following steps:

(1) Mixing the recombinant desmoglein3 with an immunologic adjuvant to prepare a vaccine taking the recombinant desmoglein3 as an antigen, wherein the immunologic adjuvant is a mixture of MPLA, addaVax or poly (I: C) and CpG 1826;

(2) Inoculating the vaccine prepared in the step (1) to a wild mouse, and inoculating according to a primary immunization mode and a boosting immunization mode; the primary immunization is to continuously inoculate 3 days of vaccine after anesthetizing the wild type mouse, and the boosting immunization is to continuously inoculate 3 days of vaccine again after 14 days of inoculation;

5. The method according to claim 4, wherein when the immunoadjuvant is MPLA, the recombinant protein vaccine is prepared according to the ratio of 3.5-50 μ g of recombinant desmoglein to 0.5-50 μ g of MPLA; when the immunologic adjuvant is a mixture of poly (I: C) and CpG1826, the recombinant protein vaccine is prepared according to the proportion of 3.5-50 mug of recombinant desmosomal mucin, 3-300 mug of poly (I: C) and 0.1-10 mug of CpG 1826; when the immunological adjuvant is AddaVax, the recombinant protein vaccine is prepared according to the volume ratio of the recombinant desmoglein3 to AddaVax of 1.