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Reorganization immunity toxin with high specificity and its preparing method

An immunotoxin and specific technology, applied in the field of recombinant immunotoxin and its preparation, can solve the problems of infection, high technical difficulty and low specificity, and achieve the effects of reducing non-specific binding, reducing toxic and side effects, and targeting specific attacks.

Inactive Publication Date: 2003-07-23
SICHUAN UNIV
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  • Abstract
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  • Claims
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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 methods, TCR blocking methods, cytokine therapy, T cell vaccination, and oral antigen-induced tolerance, etc., but these methods may be 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; ChenY, et al. Nature. 1995, 376: 177; Weiner HL, Immunology Today. ), so the treatment of autoimmune diseases is still a major clinical problem

Method used

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  • Reorganization immunity toxin with high specificity and its preparing method

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Construction of a prokaryotic expression plasmid for the preparation of recombinant immunotoxin IL-18-PE38

[0036] The base sequence of IL-18 cDNA is shown in Figure 1, the base sequence of PE38 cDNA is shown in Figure 2, and pET27 was selected as the vector. Specific steps are as follows:

[0037] 1. Mouse IL-18 gene was amplified by RT-PCR

[0038](1) One-step extraction of total RNA from activated macrophages with guanidine isothiocyanate, mixed with 10 μg of Oligo(dT), and reverse-transcribed.

[0039] (2) The primers for PCR amplification are

[0040] Upstream: 5′-CGGAATTCATAACTTTGCCGACTTCAGTGTAC

[0041] Downstream: 5′-CGGAATTCACTAACTTTGATGTAAGTTAGTGAGAG

[0042] (3) PCR reaction conditions: 0.5 minutes at 94°C, 0.5 minutes at 56°C, 1 minute at 72°C, and 10 minutes at 72°C after 30 cycles.

[0043] (4) The amplified product was analyzed by 2% agarose gel electrophoresis.

[0044] 2. The above amplified IL-18 gene fragment was recombined with the ...

Embodiment 2

[0048] Example 2: Preparation of recombinant immunotoxin IL-18-PE38

[0049] The process steps are as follows:

[0050] 1. Preparation of engineering bacteria transfected with prokaryotic expression plasmid pET27-IL-18-PE38

[0051] (1) Preparation of competent bacteria

[0052] A. Streak inoculation of Escherichia coli E.coli BL21 on the agar medium plate and culture at 37°C for 12-16 hours.

[0053] B. Take out a single colony with a size of 2-3mm from the plate, transfer it to 3ml LB medium, and culture it overnight at 37°C with shaking. The next day, take 1ml of the bacterial solution and inoculate it into a Erlenmeyer flask containing 100ml of LB medium, and culture it with strong shaking at 300rpm at 37°C for 3 hours to make the cell concentration reach 5×10 7 cells / ml, at this time, the OD of bacteria 600 Generally around 0.4.

[0054] C. Place the cultured bacteria on ice for 10 minutes, then transfer to a sterile 50ml centrifuge tube pre-chilled with ice.

[005...

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Abstract

A high-specificity recombinant immunotoxin for preventing and treating autoimmunopathy, rejection reaction and tumors is prepared from IL-18 as target moleculae and the actigve fragment PE38 of pseudomonas aeruginosa's exotoxin through configuring recombinant plasmid, preparing engineering bacteria transfected by said plasmid, expressing immunotoxin IL-18-PE38 and purifying. Its advantage is hightarget kill to TH1 cells.

Description

1. Technical field [0001] The invention relates to a recombinant immunotoxin which uses TH1 cells as target cells to specifically attack and kill activated TH1 cells and a preparation method thereof. 2. Background technology [0002] Immunotoxins (Immunotoxins, ITs) are hybrid molecules composed of targeting molecules and toxin molecules, also known as biological missiles. The earliest immunotoxins (i.e., the first generation immunotoxins) were formed by chemical coupling of carriers and toxin molecules. Such immunotoxins have large molecular weight, poor stability and uniformity, and are difficult to produce on a large scale, thus limiting their use. application. With the development of molecular biology, in recent years, people have used the recombinant technology of genetic engineering to fuse the carrier gene and the toxin gene, and then express and purify the recombinant immunotoxin. The immunotoxin produced by this new process can effectively overcome the defects of ...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00A61P37/02A61P37/06C07K16/08C07K16/16C12N15/31C12P19/34
Inventor 张林李虹李明远胡怀忠蒋忠华
Owner SICHUAN UNIV
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