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Plasmid of recombinant immunotoxin Rantes-DT390 aimed at activating Th1 cell, and its preparing method and use

An immunotoxin and recombinant plasmid technology, applied in biochemical equipment and methods, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of infection, technical difficulty, low specificity, etc., to reduce non-specific binding, reduce Toxic and side effects, the effect of reducing adverse reactions

Inactive Publication Date: 2006-05-31
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, and cannot effectively prevent the recurrence of the disease.
Therefore, researchers in related fields are seeking a specific, safe and effective treatment method from the perspective of immunotherapy: such as MHC blocking method, CD 4 Molecular blocking method, TCR blocking method, cytokine therapy, T cell vaccination, and oral antigen-induced tolerance, etc., but these methods are either due to low specificity, unstable curative effect, or technical difficulties and insurmountable toxicity. Side effects, etc. and cannot be successfully transitioned to clinical practice (see: Waldor MK, et al.Science.1985, 227: 415; Chen Y, et al. Nature. 1995, 376: 177; Weiner HL, Immunology Today. ; HolldaySD, et al.Environmental Health Perspectives.108 Suppl 3:463-73, 2000 Jun; Palmisano GL, et al.Clinical & Experimental Immunology.135(2):259-66, 2004Feb; ), thus autoimmune diseases The treatment of the disease is still a major clinical problem

Method used

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  • Plasmid of recombinant immunotoxin Rantes-DT390 aimed at activating Th1 cell, and its preparing method and use
  • Plasmid of recombinant immunotoxin Rantes-DT390 aimed at activating Th1 cell, and its preparing method and use
  • Plasmid of recombinant immunotoxin Rantes-DT390 aimed at activating Th1 cell, and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of recombinant immunotoxin eukaryotic plasmid

[0025] 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:

[0026] 1. Pretreatment of mouse Rantes gene (the following reagents were purchased from Promega Company)

[0027] (1) Use Trizol reagent to extract mouse liver total RNA according to the following steps

[0028] 1) Mouse liver tissue 100mg.

[0029] 2) Add 1ml Trizol.

[0030] 3) homogenate.

[0031] 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.

[0032] 4) Inverted and mixed for 10 times, room temperature for 5 minutes.

[0033] 5) Add 1 / 5 volume of chloroform (0.2ml, must be 1 / 5 of the total volume).

[0034] 6) Inverted and mixed for 10 times, room temperature for 5 minutes.

[0035] 7) C...

Embodiment 2

[0081] Example 2: Determination of biological activity of recombinant immunotoxin Rantes-DT390 in vitro

[0082] 1. Protein synthesis inhibition test

[0083] (1) Take the spleen of the mouse on the aseptic operating table, obtain the spleen cell suspension, and inoculate it in a cell culture bottle (about 10×10 6 cells / ml), add conA (10ug / ml) for mixed culture;

[0084] (2) After culturing for 3 days, harvest the cells, count and adjust the cell concentration to 1×10 6 cells / ml;

[0085] (3) Transfer to a 96-well cell culture plate (the culture medium is DMEM medium without leucine), and the above-mentioned transfection supernatants were divided into 1 / 2, 1 / 4, 1 / 8, 1 / 20, Add 1 / 50 diluted concentration, 50 μl / well, and set up triplicate wells for each concentration;

[0086] (4) Continue culturing for 24 hours, add 10 μl of 3H leucine (5 μci / ml), collect cells after 2 hours, perform liquid scintillation assay and calculate protein synthesis inhibition rate (relevant result...

Embodiment 3

[0092] Example 3: Preliminary therapeutic effect of recombinant immunotoxin Rantes-DT390 eukaryotic plasmid on autoimmune disease animal model EAE (experimental allergic encephalomyelitis)

[0093] 1. Establishment of EAE (Experimental Allergic Encephalomyelitis) animal model: The EAE model was established with C57BL / 6 mice according to conventional methods.

[0094](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.

[0095] (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 0.6-1.8×106 bacillus pertussis) ).

[0096] (3) In the control group, each mouse was intraperitoneally injected with 0.2 ml of a mixture of Bacillus pertussis liquid ...

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Abstract

A new type of recombinant immunotoxin plasmid for activating Th1 cells, with the chemokine Rantes as the guiding molecule and the active fragment DT390 of diphtheria toxin as the toxin molecule, constructed by gene recombination. The preparation method of the immunotoxin plasmid comprises the steps of constructing the recombinant plasmid, preparing engineering bacteria transfected by the recombinant plasmid, and the like. The immunotoxin plasmid has strict targeting, and can specifically attack activated Th1 cells with DT390 through the guidance of Rantes, and induce specific immune tolerance, thereby avoiding non-selective killing of all T cells. It is especially suitable for the prevention and treatment of autoimmune diseases and organ transplantation rejection, and can also be applied to the treatment of certain tumors.

Description

technical field [0001] The invention relates to a novel recombinant immunotoxin plasmid for specifically attacking and killing activated Th1 cells and a preparation method thereof. Background technique [0002] Immunotoxins (Immunotoxins, ITs) are hybrid molecules formed by connecting biological toxins with targeting carriers (antibodies or cytokines), also known as biological missiles or targeting toxins. The earliest immunotoxins (i.e., the first generation immunotoxins) were chemical conjugates of carriers and toxin molecules. This type of immunotoxins has strong immunogenicity, large molecular weight, poor permeability, and it is difficult to form an effective concentration and stability at the target site. And the uniformity is poor, the toxic and side effects are strong, and it is difficult to produce on a large scale, thereby limiting its application. With the continuous development of modern life sciences, people have improved immunot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/12C12N15/63C12N15/85A61K48/00A61K47/48C07K14/52
CPCA61K47/642A61P35/00A61P37/00A61P37/06C07K14/523C07K2319/55
Inventor 张林贾怡李虹陈文捷李明远
Owner ORIGISSAY BIOLOGICS TECH
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