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Anti-Pseudomonas aeruginosa exotoxin-a nanobody and its application

A nanobody and Pseudomonas aeruginosa technology, applied in the field of immunology, can solve the problems of lack of CH1 in the heavy chain, lack of binding ability of the light chain, and inability to express the CH1 domain of the heavy chain, and achieve the effect of highly specific binding activity

Active Publication Date: 2019-10-25
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

This heavy chain-only antibody may be due to mutations and deletions at the genomic level that lead to the inability to express the CH1 domain of the heavy chain, so that the expressed heavy chain lacks CH1, thereby lacking the ability to bind to the light chain, thus forming a heavy chain. chain dimer

Method used

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  • Anti-Pseudomonas aeruginosa exotoxin-a nanobody and its application
  • Anti-Pseudomonas aeruginosa exotoxin-a nanobody and its application
  • Anti-Pseudomonas aeruginosa exotoxin-a nanobody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Prokaryotic expression and purification of recombinant PE immunotoxin

[0046] 1.1 Prokaryotic expression of recombinant PE immunotoxin

[0047] The PE toxin partial sequence (AE004091.2) plasmid PE-PUC57 (synthesized by Jinweizhi Co., Ltd.) synthesized by the whole gene and the vector pET28a were cut with NcoI and NotI (purchased from NEB Company) double endonucleases at the same time, and the 1.4kb target was recovered. Gene fragments, with T 4 DNA ligase ligation, named PE-Pet28a, transformed into BL21 host bacteria, screened for kanamycin resistance, selected positive colonies, amplified and extracted plasmids, identified by NcoI and NotI double enzymes ( figure 1 : M is Trans 2K plus DNAmarker; 1 is the product of PE-Pet28a digested by NcoI and NotI). Inoculate the correctly identified strain into 200ml Kan after overnight amplification at a ratio of 1:50 + In LB medium, expand to OD at 37°C 600 When ≈0.6, add IPTG to a final concentration of 1mmol / L...

Embodiment 2

[0052] Example 2. Construction and screening of anti-PE nanobody phage display library

[0053] 2.1 Immunization of alpacas

[0054] Select a healthy adult alpaca, mix the recombinant protein PE with Freund's adjuvant at a ratio of 1:1, and immunize the alpaca with 6-7μg / Kg subcutaneous injection at multiple points on the back, and immunize the alpaca four times in total. The immunization interval was 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0055] 2.2 Isolation of camel-derived lymphocytes

[0056] Lymphocytes were analyzed 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.

[0057] 2.3 Total RNA extraction

[0058] Total RNA was extracted according to routine procedures in the technical field, and the con...

Embodiment 3

[0089] Example 3. Preparation of Anti-PE Nanobodies

[0090] 3.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL 21 (DE 3 )

[0091] Inoculate the original strain TG1 glycerolbacterium containing Nanobody nucleic acid in 5 mL of fresh LB-A medium at a ratio of 1:1000, and culture overnight at 37°C and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the instructions. After verification, transform 1 μl of the above plasmid into 100 μl competent cells, mix gently, place on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and cool in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and incubate at 37°C for 60 minutes. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 37°C overnight.

[0092] 3.2 Induced expression and extraction of nanobodies ...

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Abstract

The invention discloses a nanobody against Pseudomonas aeruginosa exotoxin A and its application. The nanobody has three unique complementarity determining regions CDR1, CDR2, and CDR3. Pseudomonas exotoxin A drug application. The anti-Pseudomonas exotoxin A nanobody provided by the present invention has specific recognition and binding ability to Pseudomonas aeruginosa exotoxin A, and can effectively neutralize the toxic effect of Pseudomonas aeruginosa exotoxin A, thereby protecting normal cells effect.

Description

technical field [0001] The invention discloses a nanobody and belongs to the field of immunology. Background technique [0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa (scientific name Pseudomonas aeruginos'), is a Gram-negative, aerobic, rod-shaped bacterium with only one-way motility. Pseudomonas aeruginosa was first isolated from wound pus by Gersard in 1882. The bacterium widely exists in water, soil, air, and animal intestines and skin in nature. It is an opportunistic pathogenic bacterium that can cause human and animal infections under certain circumstances. [0003] In 1972, P.v. Liu discovered exotoxin A, the virulence factor that causes Pseudomonas aeruginosa infection, in the culture medium of clinical isolates, and pointed out that Pseudomonas exotoxin A (PE) had necrosis on skin and mucous membranes. role, into the blood can induce sepsis. In organ tissue cells, Pseudomonas aeruginosa exotoxin A is similar to diphtheria toxin and has the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/70C12N1/21A61K39/40A61P31/00C12R1/19
CPCC12N15/70C07K16/1214A61K2039/505C07K2317/565C07K2317/569A61P31/04C07K2317/22C07K2317/76C07K2317/92
Inventor 宋海鹏刘原源于建立黄琪古一李飞周宇航王欢李靖婵
Owner 深圳市国创纳米抗体技术有限公司
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