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A fusion protein of nanobody and Pseudomonas aeruginosa exotoxin and its application

A technology of Pseudomonas aeruginosa and fusion protein, applied in the field of immunoglobulin

Active Publication Date: 2021-05-04
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the scFv of the conventional four-chain antibody, Nanobodies are comparable to their corresponding scFv in terms of affinity, but in terms of solubility, stability, resistance to aggregation, refoldability, expression yield, and DNA manipulation, library construction and ease of 3-D structure determination beyond scFv

Method used

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  • A fusion protein of nanobody and Pseudomonas aeruginosa exotoxin and its application
  • A fusion protein of nanobody and Pseudomonas aeruginosa exotoxin and its application
  • A fusion protein of nanobody and Pseudomonas aeruginosa exotoxin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of Fusion Immunotoxin

[0040] 1.1 Immunization of alpaca: Select a healthy adult alpaca, mix Pseudomonas aeruginosa exotoxin and Freund's adjuvant at a ratio of 1:1, and inject 6-7 μg / kg subcutaneously at multiple points on the back Alpacas were immunized four times in total with an interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0041] 1.2 Separation of camel-derived lymphocytes: analyze lymphocytes from the collected camel-derived anticoagulated whole blood according to routine procedures in this technical field, every 2.5×10 7 Add 1mL RNA isolation reagent to each living cell, take 1mL for RNA extraction, and store the rest at -80°C.

[0042] 1.3 Extraction of total RNA: extract total RNA according to routine procedures in this technical field, and adjust the concentration to 1 μg / μL with RNase-free water.

[0043] 1.4 Synthesis of cDNA by reverse transcript...

Embodiment 2

[0071] Example 2 Cytotoxicity Experiment of Fusion Immunotoxin

[0072] 2.1 Quantitative detection of MMP enzymes in SW480 and LoVo cells: ready 25cm 2 Cultured cells to be tested in cell culture flasks or 60mm cell culture dishes (1~5×10 6 1), carefully add 3mL Reagent A, cover the growth surface, carefully remove the cleaning solution, and gently scrape off the cells with a cell scraper. Add 3mL Reagent A, mix the cells, transfer to a pre-cooled 15mL conical centrifuge tube, centrifuge at 4°C for 5min at a speed of 300g, and carefully remove the supernatant. Add 500 μL Reagent B, mix thoroughly, transfer to a pre-cooled 1.5mL centrifuge tube, vortex vigorously for 15 seconds, and incubate in an ice bath for 30 minutes. Centrifuge for 5 minutes in a micro-tabletop centrifuge at 4°C at a speed of 13 000 rpm. Carefully pipette 500 μL of the supernatant into a new pre-cooled 1.5mL centrifuge tube, pipette 10 μL for protein quantitative detection, and immediately store at -70°...

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Abstract

The invention discloses a fusion protein of a nanobody and Pseudomonas aeruginosa exotoxin. The fusion protein is formed by cascading the nanobody with targeting function or its variable region fragment and the Pseudomonas aeruginosa exotoxin. The invention also provides the application of the fusion protein in the preparation of drugs for treating tumors. The fusion protein has good specificity and killing effect on tumor antigens, has protective effect on normal tissue cells, and has good clinical application prospect.

Description

technical field [0001] The invention discloses a fusion protein and its immunological application, and belongs to the technical field of polypeptides, more specifically, to the technical field of immunoglobulins. Background technique [0002] Tumor is an important factor that seriously endangers human health. Both traditional chemotherapy drugs and radiotherapy have poor targeting characteristics, so the development of targeted drugs has become a research hotspot in the treatment of malignant tumors. The initial targeted therapy for tumor cells uses unlabeled monoclonal antibodies, the purpose is to induce cell death or use complement-mediated cytotoxicity to kill target cells, but cancer cells usually show anti-apoptosis. function, and it can also frequently modulate antigens to avoid the damage caused by the above two killing mechanisms. Subsequently, radioimmunotherapy technology appeared, which not only can increase cytotoxicity, but also relies on the mechanism of ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K38/16A61K47/68A61P35/00
CPCA61K38/00A61P35/00C07K14/21C07K16/3007C07K2317/569C07K2319/33C07K2319/55
Inventor 宋海鹏于建立刘原源黄琪李飞古一周宇杭
Owner 深圳市国创纳米抗体技术有限公司
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