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Plasmid of recombinant immunotoxin IP 10-DT 390 aimed at activating Th1 cell, and its preparing method and use

An immunotoxin and recombinant plasmid technology, applied in recombinant DNA technology, drug combination, pharmaceutical formulations and other directions, can solve the problems of inability to effectively prevent disease recurrence, technical difficulties, toxic side effects, and unstable curative effects, and avoid the preparation process and It is difficult to standardize, reduce toxic and side effects, and is easy to industrialize.

Inactive Publication Date: 2006-06-28
ORIGISSAY BIOLOGICS TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current treatment measures mainly use various non-specific immunosuppressants, but the immune system of patients is often suppressed generally, leading to infection, tumor occurrence and bone marrow suppression, etc., and the recurrence of the disease cannot be effectively prevented.
Therefore, researchers in related fields are prompted to seek a specific, safe and effective treatment method from the perspective of immunotherapy: such as TCR blocking method, cytokine therapy, MHC blocking method, CD4 molecular blocking method, T cell vaccination and Oral antigens induce tolerance, etc., but these methods cannot be successfully transitioned to the clinic due to low specificity, unstable curative effect, technical difficulty, and insurmountable toxic side effects (see: Waldor MK, et al. Science. 1985, 227: 415; Chen Y, et al. Nature. 1995, 376: 177; Weiner HL, Immunology Today. 1997, 18: 355; Hollday SD, et al. Environmental Health Perspectives. 108 Suppl 3: 463-73 , 2000 Jun; PalmisanoGL.et al.Clinical & Experimental Immunology.135(2):259-66, 2004Feb), so the treatment of autoimmune diseases is still a major clinical problem

Method used

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  • Plasmid of recombinant immunotoxin IP 10-DT 390 aimed at activating Th1 cell, and its preparing method and use
  • Plasmid of recombinant immunotoxin IP 10-DT 390 aimed at activating Th1 cell, and its preparing method and use
  • Plasmid of recombinant immunotoxin IP 10-DT 390 aimed at activating Th1 cell, and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of recombinant immunotoxin eukaryotic expression plasmid

[0024] The vector used was the SRα eukaryotic plasmid containing DT390 (provided by PhD. Hu Huaizhong, University of Wisconsin, USA, Invitrogen Company), and the specific steps were as follows:

[0025] 1. Pretreatment of mouse IP10 gene (the following reagents were all purchased from Promega Company)

[0026] (1) Use Trizol reagent to extract mouse liver total RNA according to the following steps

[0027] 1) Mouse liver tissue 100mg.

[0028] 2) Add 1ml Trizol.

[0029] 3) homogenate.

[0030] The homogenate should be thorough, and then transferred to EP tubes. When the amount of tissue homogenate is > 100mg, it will be divided into 1ml / each EP tube.

[0031] 4) Inverted and mixed for 10 times, room temperature for 5 minutes.

[0032] 5) Add 1 / 5 volume of chloroform (0.2ml, must be 1 / 5 of the total volume).

[0033] 6) Inverted and mixed for 10 times, room temperature for 5 minutes...

Embodiment 2

[0079] Example 2: Determination of biological activity of recombinant immunotoxin IP10-DT390 in vitro

[0080] 1. Determination of cytotoxicity by flow cytometry:

[0081] (1) Take the mouse spleen on a sterile operating table, make a spleen cell suspension with RPMI1640 medium, and mix it with conA (10ug / ml);

[0082] (2) After culturing for 3 days, add the above-mentioned transfection supernatant according to the concentrations diluted by 1 / 2, 1 / 4, 1 / 8, 1 / 20, and 1 / 50 of the stock solution, 50 μl / well, and set up triplicate wells for each concentration;

[0083] (3) Continue culturing for 24 hours, collect the cells, and measure the cytotoxicity (CD4 + : T cell membrane surface marker; IFN-γ: Th1 intracellular marker; IL-4: Th2 intracellular marker; CD19 + : B cell membrane surface marker);

[0084] (4) The results of flow cytometry showed that the number of T cells and Th1 cells decreased by 40%-60%, but there was no such effect on Th2 and B cells (such as figure 2 sho...

Embodiment 3

[0091] Example 3 Preliminary therapeutic effect of recombinant plasmids on autoimmune disease animal model EAE (experimental allergicencephalomyelitis)

[0092] 1. Establishment of EAE (Experimental Allergic Encephalomyelitis) animal model: C57BL / 6 mice were immunized with MBP according to conventional methods to establish an EAE model.

[0093] (1) Use C57BL / 6 mice to establish an EAE animal model, mix the self-extracted MBP crude extract with FCA (containing Mycobacterium tuberculosis 5mg / ml) in equal volumes, use a 3ml syringe to repeatedly push and pull to form a water-in-oil emulsion , by intraperitoneal injection.

[0094] (2) Immunization dose: each mouse was injected with 0.4ml of MBP: FCA (1:1) mixed solution, 0.2ml of pertussis bacilli liquid: PBS (1:50) mixed solution (containing 0.6-1.8×10 6 indivual).

[0095] (3) In the control group, each mouse was intraperitoneally injected with 0.2ml of a mixture of Bacillus pertussis liquid and PBS (containing 0.6-1.8×10 6...

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Abstract

The high efficiency expression plasmid of recombinant immune toxin IP10-DT390 constituted by means of gene engineering technology is for activated Th1 cell specifically. After transfected into body, the immune toxin plasmid expresses high specific recombinant immune toxin with IP10 as targeting molecule, active diphtherin segment DT390 as toxin molecule and the specific membrane acceptor CXCR3 of activated Th1 cell as the target point. The preparation process of the immune toxin plasmid includes the constitution of recombinant plasmid, the preparation of recombinant plasmid transfected engineering bacterium and other steps. The plasmid expressed recombinant immune toxin has strict targeting property of attacking activated Th1 cell specifically by means of targeting IP10 to induce specific immunity tolerance and avoid killing normal T cells, and is especially suitable for preventing and treating of autoimmune disease and organ transplantation rejection, and some tumors.

Description

technical field [0001] The invention relates to a novel recombinant immunotoxin plasmid expressing IP10 as a guiding molecule and DT390, an active fragment of diphtheria toxin as a toxin molecule, specifically attacking and killing activated Th1 cells and a preparation method thereof. Background technique [0002] Immunotoxins (Immunotoxins, ITs) are targeting drugs that connect biological toxins with antibodies or cytokines, also known as biomissiles or targeting toxins. The first generation of immunotoxins is formed by chemical coupling of carriers and toxin molecules. Such immunotoxins have disadvantages such as poor stability, large molecular weight, strong immunogenicity, poor permeability, and poor curative effect, and are difficult to produce on a large scale. Therefore, limit its application. With the further development of genetic engineering recombination technology, the carrier gene is fused with the toxin gene, and the recombinant ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63A61K48/00A61P35/00
Inventor 张林陈文捷李虹贾怡李明远
Owner ORIGISSAY BIOLOGICS TECH
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