Method for constructing pemphigus vulgaris animal model

A pemphigus vulgaris and construction method technology, applied in animal husbandry and other fields, can solve problems such as high cost, limited application of immunotherapy, and no PV animal model.

Active Publication Date: 2022-07-29
HOSPITAL OF DERMATOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this model requires gene knockout mice and immunodeficiency mice, and the cost is high, and the immunodeficiency of recipient mice limits its application in immunotherapy and vaccine development
[0004] Active immunization induction is an important method for animal models of autoimmune diseases, and it is widely used in the experimental research of autoimmune diseases such as multiple sclerosis and arthritis, but there is no report of PV animal models induced by active immunization

Method used

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  • Method for constructing pemphigus vulgaris animal model
  • Method for constructing pemphigus vulgaris animal model
  • Method for constructing pemphigus vulgaris animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 MPLA as an immune adjuvant recombinant protein vaccine induces a PV model.

[0039] 1. Experimental materials

[0040] SPF grade 7-week female C57BL / 6J mice, recombinant Dsg3 protein solution (Nanjing Mingyan), MPLA (invivogen), 4% paraformaldehyde (Biosharp), OCT embedding medium (Sakura), goat anti-mouse IgG-FITC, Goat anti-mouse IgG-HRP (Abclonal), etc.

[0041] 2. Experimental method

[0042] 1. Vaccination of mice

[0043] Prepare recombinant protein vaccine. After the mice were anesthetized, 50 μl of recombinant protein vaccine (containing 5 μg of Dsg3 protein and 5 μg of MPLA) was subcutaneously inoculated through the soles of the feet for 3 consecutive days. On the 14th day, the mice were boosted and vaccinated in the same way for 3 days.

[0044] 2. Rat tail, sole and back skin tissue extraction and histological examination

[0045] On day 35, mice were sacrificed by cervical dislocation after anesthesia. Use a sharp scalpel and ophthalmic scis...

Embodiment 2

[0052] Example 2 Poly(I:C) and CpG1826 as immune adjuvant recombinant protein vaccine induced PV model.

[0053] 1. Experimental materials

[0054] SPF grade 7-week female C57BL / 6J mice, recombinant Dsg3 protein solution (Nanjing Mingyan), poly(I:C) (invivogen), CpG1826 (Sangong), 4% paraformaldehyde (Biosharp), OCT embedding medium (Sakura), goat anti-mouse IgG-FITC, goat anti-mouse IgG-HRP (Abclonal), etc.

[0055] 2. Experimental method

[0056] 1. Vaccination of mice

[0057] Recombinant protein vaccine was prepared. After anesthesia, mice were subcutaneously inoculated with 50 μl recombinant protein vaccine (including Dsg3 protein 5 μg, poly(I:C) 30 μg, and CpG1826 1 μg) for 3 consecutive days. On the 14th day, mice were boosted and vaccinated in the same way for 3 days.

[0058] 2. Rat tail, sole, back skin tissue extraction and histological examination

[0059] On day 35, mice were sacrificed by cervical dislocation after anesthesia. Use a sharp scalpel and ophtha...

Embodiment 3

[0066] Example 3 AddaVax is an immune adjuvant recombinant protein vaccine induced PV model.

[0067] 1. Experimental materials

[0068] SPF grade 7-week female C57BL / 6J mice, recombinant Dsg3 protein solution (Nanjing Mingyan), AddaVax adjuvant (invivogen), goat anti-mouse IgG2c-HRP (Southern Biotech), etc.

[0069] 2. Experimental method

[0070] 1. Vaccination of mice

[0071] The recombinant protein vaccine was prepared according to the ratio of Dsg3 protein and AddaVax volume to 1:1. After the mice were anesthetized, 50 μl of recombinant protein vaccine (containing 5 μg of Dsg3 protein and 25 μl of AddaVax) was subcutaneously inoculated through the soles of the feet for 3 consecutive days. On the 14th day, the mice were boosted and vaccinated in the same way for 3 days.

[0072] 2. ELISA assay

[0073] Serum was prepared at different time points, aliquoted and frozen for ELISA. Prepare 50 μg / ml Dsg3 solution, add 100 μl / well to the ELISA plate, coat overnight at 4°C...

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Abstract

The invention discloses a pemphigus vulgaris animal model construction method, which comprises the following steps: carrying out primary immunization/booster immunization on a wild type mouse by using a vaccination technology and taking desmoplasmin 3 as a vaccine antigen, starting to appear phenotypes within 28-35 days, including blisters, epidermis exfoliation and skin ulceration on the rear feet and the tail of the mouse with the overall phenotype; a serum autoantibody; the pemphigus vulgaris immunotherapy kit has the advantages that pemphigus vulgaris changes such as epidermis spinous layer debonding and antibody deposition in histological examination are simulated, part of mice have disease phenotypes such as local skin unhairing, fester and canthus conjunctivitis, clinical symptoms of pemphigus vulgaris are simulated, and an important tool is provided for pemphigus vulgaris immunotherapy.

Description

technical field [0001] The invention belongs to the technical field of disease animal model construction methods, in particular to the construction of an animal model of pemphigus vulgaris. Background technique [0002] Pemphigus (pemphigus) is a class of autoantibody-mediated, life-threatening autoimmune skin diseases, including pemphigus vulgaris (PV), pemphigus foliaceus (pemphigus foliaceus), Tumor pemphigus (paraneoplastic pemphigus) and so on [1-3]. There are autoreactive plasma cells in PV patients, and the autoantibodies secreted and produced bind to the surface adhesion protein desmoglein3 (Dsg3) of skin epidermal cells, resulting in acantholysis, the formation of skin blisters, repeated ulcers and other symptoms . Damage to the skin barrier not only brings pain and increases the chance of pathogen infection, but also affects the appearance and increases the psychological burden of patients. The development of animal models that can simulate clinical symptoms and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027
CPCA01K67/027A01K2207/10A01K2207/05A01K2207/20A01K2227/105A01K2267/0325
Inventor 陆前进高长醒刘梅张博辛月王来尹昊媛
Owner HOSPITAL OF DERMATOLOGY CHINESE ACAD OF MEDICAL SCI
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