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Recombinant immunotoxin gigantoxin-4-4d5 scFv and preparation method and use thereof

A technology of immunotoxin and cytolysin, which is applied in the field of genetic engineering and achieves the effects of good application prospect, obvious anti-tumor effect and low production cost

Inactive Publication Date: 2019-07-12
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, there is no report on the use of anti-human epidermal growth factor receptor 2 single-chain antibody 4D5 scFv fused with sea anemone cytolysin Gigantoxin-4 to treat tumors

Method used

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  • Recombinant immunotoxin gigantoxin-4-4d5 scFv and preparation method and use thereof
  • Recombinant immunotoxin gigantoxin-4-4d5 scFv and preparation method and use thereof
  • Recombinant immunotoxin gigantoxin-4-4d5 scFv and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of recombinant immunotoxin Gigantoxin-4-4D5scFv

[0037] This embodiment 1 is implemented under the following conditions of implementation and technical requirements:

[0038] (1) Construction of pET28a(+) / SUMO-Gigantoxin-4-4D5 scFv recombinant plasmid

[0039] A. Cloning of SUMO-Gigantoxin-4-4D5 scFv gene fragment. The primers designed according to each fragment template and the linker(G4S)3 to be added are shown in Table 1:

[0040] Table 1

[0041]

[0042] Use C1 and C2, C3 and B2, B3 and B4 to PCR out the corresponding fragments with SUMO, Gigantoxin-4, and 4D5scFv as templates respectively. The gene of linker (G4S)3 has been designed into the primers of B2 and B3. The PCR conditions are: 98°C Pre-denatured for 1 min, denatured at 98°C for 10s, annealed at 58°C for 30s, extended at 72°C for 30s, cycled 35 times, and finally extended at 72°C for 7min. The PCR products were identified by agarose gel, and the respective fragments of intere...

Embodiment 2

[0056] Example 2 Activity Detection of Recombinant Immunotoxin Gigantoxin-4-4D5 scFv

[0057] 1. The inhibitory effect of Gigantoxin-4-4D5 scFv on the proliferation of ovarian cancer cell SK-OV-3:

[0058] MTT method was used to detect the inhibitory effect of recombinant immunotoxin Gigantoxin-4-4D5 scFv on the proliferation of ovarian cancer cell SK-OV-3, and normal human embryonic kidney cell HEK-293 was used as a control. The specific operation was as follows: 0.25% trypsin solution Digest SK-OV-3 and HEK-293 to make a single-cell suspension, inoculate 8000 cells per well in a 96-well plate, each well volume is 100uL, fill the edge wells with sterile PBS, and store at 37°C for 5 %CO 2 Cultivate in the incubator for 16h. Dilute Gigantoxin-4-4D5 scFv with medium to 0.027, 0.054, 0.108, 0.216, 0.432nM, absorb the original medium, and add 100uL of medium containing different concentrations of Gigantoxin-4-4D5scFv, each concentration has 5 parallel, the control group is the ...

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Abstract

The invention discloses recombinant humanized immunotoxin Gigantoxin-4-4D5scFv. The immunotoxin contains anti human epidermal growth factor receptor-2 (HER-2) single-chain antibody 4D5scFv and sea anemone cytolysin Gigantoxin-4 and is formed through fusing and expression of HER-2 and sea anemone cytolysin Gigantoxin-4. Targeting of the single-chain antibody at a HER-2 high-expression tumor and the splitting effect of Gigantoxin-4 on cytomembrane are used, so that a specific killing effect on the tumor is achieved. The immunotoxin has a strict targeting property, and damage to normal cells is avoided; the effect of tumor killing can be achieved without cell internalization and transferring; drug resistance cannot be produced easily; a preparation method is simple, and the antitumous effect is obvious. The immunotoxin has a good application prospect and can be used for treating high-expression HER-2 tumor cells.

Description

technical field [0001] The invention relates to genetic engineering technology, in particular to a recombinant immunotoxin Gigantoxin-4-4D5 scFv and its preparation method and application. Background technique [0002] Malignant tumors have become one of the most important diseases threatening human health, especially in my country, where tumors have surpassed cardiovascular diseases to become the number one killer causing the most deaths. [0003] At present, chemotherapy and radiotherapy are the main methods for treating tumors, but conventional radiotherapy and chemotherapy basically do not have the specificity for tumors. Radiotherapy and chemotherapy destroy cancer cells and also have a killing effect on normal cells, thus causing great toxic side effects on the human body. At the same time, it will also lead to the destruction of the host immune system. After radiotherapy and chemotherapy, it is easy to cause various complications and tumor recurrence. The characterist...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/16C12N15/62C12N15/70C12N1/21C12P21/00C12P21/06A61K38/17A61K47/68A61P35/00
Inventor 孙爱友吕欣欣魏东芝
Owner EAST CHINA UNIV OF SCI & TECH
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