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Plasmid containing recombinant immunological toxin MIP-1ª‡-DT390 aiming at activated Th1 cell, its preparation method and uses

A technology of immunotoxin and recombinant plasmid, applied in the direction of recombinant DNA technology, drug combination, pharmaceutical formula, etc., can solve the problem of ineffective prevention of disease recurrence, avoid preparation process and difficulty in standardization, relieve clinical symptoms, and have good therapeutic effect Effect

Inactive Publication Date: 2006-05-03
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 non-specific immunosuppressants, which often cause the patient's immune system to be generally suppressed, leading to infection, tumor occurrence and bone marrow suppression, etc., and cannot effectively prevent the recurrence of the disease

Method used

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  • Plasmid containing recombinant immunological toxin MIP-1ª‡-DT390 aiming at activated Th1 cell, its preparation method and uses
  • Plasmid containing recombinant immunological toxin MIP-1ª‡-DT390 aiming at activated Th1 cell, its preparation method and uses
  • Plasmid containing recombinant immunological toxin MIP-1ª‡-DT390 aiming at activated Th1 cell, its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of eukaryotic expression plasmids for recombinant immunotoxins

[0031] The vector is SRα eukaryotic plasmid containing DT390 (provided by PhD. Hu Huaizhong, University of Wisconsin, USA, and can be purchased from Invitrogen Company), and the specific steps are as follows:

[0032] 1. Obtain mouse MIP-1α gene

[0033] (1) Extract mouse liver total RNA with Trizol reagent (purchased from Invitrogen):

[0034] 1) Take 100 mg of mouse liver tissue under sterile conditions.

[0035] 2) Add 1ml Trizol.

[0036] 3) Homogenate (should be thorough, then transfer to EP tubes, when tissue homogenate volume > 100mg, pack separately, 1ml / each EP tube).

[0037] 4) Mix by inverting for 10 times, and let stand at room temperature for 5 minutes.

[0038] 5) Add 1 / 5 volume (0.2ml) of chloroform (must be 1 / 5 of the total volume).

[0039] 6) Mix by inverting for 10 times, and let stand at room temperature for 5 minutes.

[0040] 7) Centrifuge at 12000g for ...

Embodiment 2

[0086] Example 2: Determination of biological activity of recombinant immunotoxin MIP-1α-DT390 in vitro

[0087] 1. Cytotoxic effect of MIP-1α-DT390 in transfection supernatant on effector cells (MTT method):

[0088](1) Preparation of effector cells: 6-8 weeks old, about 20g female C57BL / 6 mice were killed after eyeball bleeding, aseptically dissected and stripped the spleen, gently pressed the spleen tissue with a syringe needle, added RPMI1640 medium, collected and centrifuged, Obtain a suspension of splenocytes and adjust the cell concentration to 5 x 10 6 / ml, inoculated in a cell culture flask, added RPMI1640 medium containing concanavalin A (ConA) (final concentration of ConA was 5 μg / ml), cultivated in a 5% CO2 incubator at 37°C, and cultivated for 72 hours Afterwards, ConA-activated lymphocytes grown in suspension were collected as effector cells.

[0089] (2) Adjust the collected effector cell concentration to 1×10 6 / ml, add to 96-well cell culture plate, 100μl / w...

Embodiment 3

[0098] Example 3: Preliminary therapeutic effect of eukaryotic plasmid of recombinant immunotoxin MIP-1α-DT390 on autoimmune disease animal model EAE

[0099] 1. Establishment of EAE animal model: C57BL / 6 mice were immunized with myelin basic protein (MBP) according to a conventional method to establish an EAE model.

[0100] (1) C57BL / 6 mice were used to establish an EAE animal model, and the self-extracted MBP crude extract was mixed with complete Freund adjuvant (full freund adjuvant, 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, and use the method of intraperitoneal injection.

[0101] (2) Immunization dose: each mouse was injected with 0.4ml of MBP:FCA (1:1) mixture, 0.2ml of pertussis bacterial liquid:PBS (1:50) mixture (containing 0.6-1.8×10 6 indivual).

[0102] (3) In the control group, each mouse was intraperitoneally injected with 0.2ml of a mixture of Bacillus p...

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Abstract

In the invention, selecting MIP-1alpha as guide molecule, active segment DT390 of diphtheria toxin as toxin molecule, constructing eukaryon plasmid of MIP-1alpha-DT390 recombination immunotoxin, and expressing the product in transfection animal body. The experiments show: the recombination immunotoxin MIP-1alpha-DT390 has character to special attack and kill activated Th1 cell while no effect to Th2 and B cells. After curing by the product, the clinical symptom of EAE is released obviously; the pathological section shows that compared with un-cured group, in the cured group, the cerebellum and spinal demyelination lesions reduces, meninges and vein peripheral lymphocyte infiltration decreases.

Description

technical field [0001] The invention relates to a recombinant immunotoxin expression eukaryotic plasmid for specifically attacking and killing activated Th1 cells, a preparation method and application thereof. Background technique [0002] Immunotoxins (Immunotoxins, ITs) are drugs that link biological toxins with targeting molecules (antibodies or cytokines), also known as biological missiles or targeting toxins. The first-generation immunotoxins are produced by chemical coupling of carriers and toxin molecules. They have large molecular weight, strong immunogenicity, poor stability, short half-life, poor permeability, and poor curative effect. They are difficult to produce on a large scale, which limits their application. In recent years, people have improved immunotoxins through genetic recombination technology. The targeting carrier began to use genetically engineered antibodies and some ligands, which reduced the molecular weight without affectin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62A61K48/00A61P37/00
Inventor 张林吕梅励李虹陈文捷贾怡梁伟波李明远
Owner ORIGISSAY BIOLOGICS TECH
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