Antigen capable of increasing CD4 + CD25 + Foxp3 + regulatory T cells and application thereof

A foxp3, regulatory technology, applied in the field of immunology, can solve the problems of immediate rebound of autoimmune response, side effects of patients, and relapse of disease, and achieve the effect of inhibiting inflammatory symptoms and immune pathological reactions, and has broad application prospects.

Inactive Publication Date: 2010-12-22
NANJING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But some of these drugs are not only expensive and cause unbearable side effects for patients, but also on

Method used

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  • Antigen capable of increasing CD4 + CD25 + Foxp3 + regulatory T cells and application thereof
  • Antigen capable of increasing CD4 + CD25 + Foxp3 + regulatory T cells and application thereof
  • Antigen capable of increasing CD4 + CD25 + Foxp3 + regulatory T cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Sequence alignment analysis of SjHSP60 homology, crossover epitope analysis, and detection of its natural expression.

[0059]1. According to the full-length cDNA sequence of the SjHSP60 gene (sequence number: AY813151) in Genebank, primers were designed in advance: the upstream primer was 5'-cgcggatcccaaccggtgacaatgttacgag-3' (including the BamHI restriction site and the initial codon ATG), and the downstream primer was It is 5'-ccgctcgagattaagagcaggcagtgtttac-3' (containing XholI restriction site and stop codon TAA), synthesized by Shanghai Invitrogen Trading Co., Ltd. (Invitrogen); the full-length SjHSP60 ORF was amplified from the total cDNA of Schistosoma japonicum by PCR The sequence was sent to a biotechnology company (Shanghai Yingwei Jieji Trading Co., Ltd., Invitrogen) for sequencing, and the PCR product was recovered; the sequencing result was shown in SEQ.ID.NO.2, and then passed the online biological tool Biology WorkBench ( http: / / workbench.sds...

Embodiment 2

[0067] Example 2: SjHSP60 immunized mice in vivo or stimulated mouse lymphocytes in vitro, and observed the effect of SjHSP60 on CD4 + CD25 + Foxp3 + Effect of Tregs.

[0068] 1. With the SjHSP60 purified in Example 1 (3), filter the bacteria and remove the toxoid and related molecules therein through Polymyxin B-Agarose (Sigma, P1411), and then pass through the Limulus amebocyte lysate (LAL) detection kit E -TOXATE Kits (Sigma, ET0200), verify the endotoxin removal effect of the purified protein, 6 Lymphocytes were stained and marked according to the operation manual of the MouseRegulatory T Cell Staining Kit (eBioscience, 88-8111-40), and CD4 was detected by flow cytometer FACSCalibur. + CD25 + Foxp3 + Ratio of Tregs.

[0069] 2. Separate the spleen and lymph nodes of normal BALB / c mice, and prepare a single lymphocyte suspension according to the above (1); after counting the cells, transfer them to 96-well round-bottom culture plates, 5×10 5 cells / well; and then stimu...

Embodiment 3

[0071] Example 3: To investigate whether SjHSP60 can inhibit inflammation and immunopathological responses in an inflammation model.

[0072] 1. According to the method in Example 2 (1), divide into two groups: SjHSP60 group / PBS group, and in vivo immunize DBA1 mice (Nanjing University National Genetic Engineering Mouse Resource Bank, 6-8 weeks old, female). Establish collagen-induced arthritis (CIA) mouse model: mix bovine collagen type II (CII) Bovine Collagen Type II (BD, 354257) and Mycobacterium tuberculosis H37RA (Difco, 3114), and dilute with 1×PBS to make the concentration of the two 2mg / ml, 3mg / ml respectively; add an equal volume of incomplete Freund's adjuvant CFA to the mixed system, mix on a vortex shaker for 2min, repeat 6-8 times, once every 30min; One week after SjHSP60 / PBS, the tails of the two groups of mice were subcutaneously immunized with the mixed antigen to induce CIA, 100ul / mouse. One week after the induction, the two groups of mice were immunized wit...

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Abstract

The invention belongs to the immunology field, in particular to a proteantigen molecule-Japanese blood fluke heat shock protein 60KDa (SjHSP60) which is derived from a blood fluke and is capable of increasing CD4 + CD25 + Foxp3 + regulatory T cells and application thereof. The SjHSP60 has a full-length amino acid sequence as shown in SEQ.ID.NO.1, has a series of identical or highly similar cross-reactive T cell epitopes with HSP60 infected by a host. After being used for mouse in vivo immunization or in vitro stimulus to mouse spleen and lymph gland cells, the SjHSP60 can obviously increase CD4 + CD25 + Foxp3 + Tregs. In practical application, the SjHSP60 can effectively relieve inflammatory symptoms and immunopathological effects caused by arthritis, thereby having wide prospects in the aspects of immunological suppression inducement and treatment of immunological diseases.

Description

technical field [0001] The invention belongs to the field of immunology, in particular to a method for increasing CD4 + CD25 + Foxp3 + Schistosoma japonicum heatshock protein 60KDa (Schistosoma japonicum heatshock protein 60KDa, SjHSP60), an antigenic molecule derived from schistosome of regulatory T cells (Tregs), and its application. Background technique [0002] Around the 1990s, developed countries in the West put forward the "hygiene hypothesis", arguing that in developed countries where the sanitation environment has been greatly improved, the incidence of some allergic diseases and autoimmune diseases has greatly increased. Improvement is related to the reduction of chances of pathogen infection in the population (Wills-Karp, et al, Nat Rev Immunol, 2001, 1:69-75). In the following years, scientists have proved through some animal models of allergic diseases and autoimmune diseases that chronic infection of some pathogens can indeed prevent the occurrence of these ...

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K38/17A61P37/02A61P37/06A61P29/00
CPCA61K38/1709C07K14/43559A61P29/00A61P37/02A61P37/06
Inventor 苏川周莎汪雪峰贺蕾刘丰
Owner NANJING MEDICAL UNIV
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