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P19/Ebi3 compound and application of polyclonal antibody of P19/Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment

A complex and antibody technology, applied in the direction of antibodies, antibody medical components, medical preparations containing active ingredients, etc., can solve the problems of poor efficacy and obvious adverse reactions in patients

Inactive Publication Date: 2015-11-25
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional method of treating systemic lupus erythematosus is to use antimalarial drugs, anti-inflammatory drugs and immunosuppressive drugs. When the symptoms are difficult to control, corticosteroids are supplemented to non-steroidal anti-inflammatory agents; although in controlling the disease and preventing the development of the disease It has a significant effect, but most of them have obvious adverse reactions, and the curative effect is not good for some patients

Method used

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  • P19/Ebi3 compound and application of polyclonal antibody of P19/Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment
  • P19/Ebi3 compound and application of polyclonal antibody of P19/Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment
  • P19/Ebi3 compound and application of polyclonal antibody of P19/Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Human IL-12 family cytokine subunits P19 and Ebi3 can form a P19 / Ebi3 complex

[0072] 1. Materials

[0073] Human Ebi3 protein was purchased from Origene (Cat. No. TP723769), human P19 protein was purchased from Origene (Cat. No. TP309680), anti-Ebi3 antibody was purchased from Santa Cruz Biotech (Cat. No. sc-32869), anti-P40 antibody was purchased from Santa Cruz Biotech (Cat. No. sc-7926 ), anti-P19 antibody was purchased from Santa Cruz Biotech (product number sc-50303), anti-c-Jun antibody was purchased from Santa Cruz Biotech company (product number sc-44)

[0074] 2. Method

[0075] 1. Immunoprecipitation

[0076] 1) Agrose-protein A / G cleaning: Take 60 μl Agrose-protein A / G into EP tube (scissor tip), add 500 μl pre-cooled PBS, centrifuge at 4°C, 5000 rpm for 2 min (wash twice).

[0077] 2) Antibody and Agrose-protein A / G pre-incubation: Add 1 μg of anti-Ebi3 antibody, anti-p40 antibody, anti-P19 antibody, and anti-c-Jun antibody to 15 μl of washed ...

Embodiment 2

[0108] Example 2 Mouse IL-12 family cytokine subunits P19 and Ebi3 can form a P19 / Ebi3 complex

[0109] 1. Materials

[0110] Plasmids and Strains:

[0111] P19 / pReceiver-Lv18, Ebi3 / pEZ-LV122X expression plasmid, JM109 strain

[0112] 2. Method

[0113] 1. Primer design

[0114] Amplify the P19ORF primer in the P19 / Ebi3 complex

[0115] Sense strand: 5'-ATGCTGGATTGCAGAGCAGT-3' (SEQ ID NO: 5)

[0116] Antisense strand: 5'-GTAAGCTGTTGGCACTAAGG-3' (SEQ ID NO: 6)

[0117] Sense strand after adding EcoRI site and protection base:

[0118] 5'-CCGGAATTCATGCTGGATTGCAGAGCAGT-3' (SEQ ID NO: 7)

[0119] Antisense strand after adding XhoI site and protective base:

[0120] 5'-CCGCTCGAGGTAAGCTGTTGGCACTAAGG-3' (SEQ ID NO: 8)

[0121] Primers for amplifying the Ebi3 ORF in the P19 / Ebi3 complex

[0122] Sense strand: 5'-ATGTCCAAGCTGCTCTTCCT-3' (SEQ ID NO: 9)

[0123] Antisense strand: 5'-TCAGGGCTTATGGGGTGCAC-3' (SEQ ID NO: 10)

[0124] Sense strand after adding EcoRI site and prot...

Embodiment 3

[0177] Example 3 Purification and mass spectrometry identification of mouse P19 / Ebi3 complex

[0178] 1. Method

[0179] 1. Co-expression of mouse Ebi3-Flag / P19-His

[0180] Experimental process with reference to embodiment 2.

[0181] 2. Purification of Ebi3-Flag / P19-His

[0182] 1) Nickel column treatment

[0183] Aspirate the 1ml nickel column into the empty chromatographic tube, and add 3ml of equilibration buffer after the liquid in it flows out.

[0184] 2) Add 10ml of the supernatant to the equilibrated nickel column, and repeat the loading of the sample passed through the column 3 times, and take 20 μl of the sample passed through the column for gel running.

[0185] 3. Coomassie brilliant blue staining

[0186] 1) The purified protein was subjected to SDS-PAGE, and the experimental process was as described in Example 1. Soak the gel in fixative for at least 30 min.

[0187] 2) Soak the fixed gel in hot staining solution (dissolve 0.29g Coomassie Brilliant Blue ...

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Abstract

The invention discloses a P19 / Ebi3 compound and an application of a polyclonal antibody of the P19 / Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment. Experiments prove that compared with normal mice, mice with SLE have high-level expression of the P19 / Ebi3 compound, with the application of the polyclonal antibody resistant to P19 / Ebi3 protein, the expression level of antibodies and the number of immune cells in the mice can be reduced, and SLE can be treated through preparation of the antibodies resistant to P19 / Ebi3, so that the P19 / Ebi3 compound and an inhibitor thereof are used for preparing drugs for diagnosing and treating SLE and have excellent development prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to the application of P19 / Ebi3 complex and its polyclonal antibody in the diagnosis and treatment of systemic lupus erythematosus. Background technique [0002] Systemic Lupus Erythematosus (SLE) is an autoimmune disease that seriously threatens human health and life, with high mortality and disability rates. Its etiology and pathogenesis are still unclear, but it is generally believed that it is the result of environmental factors acting on the body of a certain genetic background (including different types of expression of histocompatibility, cytokines, cytokine receptors, etc.). Its immunopathological feature is the production of a variety of high and low levels of pathogenic autoantibodies in the body, and lymphocyte apoptosis at the same time. A large number of nuclear contents and ribosomes are released to become self-antigens; antigens and antibodies c...

Claims

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Application Information

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IPC IPC(8): C07K14/54C12P21/02C07K16/24G01N33/53A61K39/395A61P37/02
CPCA61K39/39516C07K14/54C07K16/244G01N33/53
Inventor 王仁喜肖鹤韩根成陈国江黎燕沈倍奋侯春梅王小茜魏寅祥刘晓玲张玉
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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