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Neutralizing monoclonal antibodies against B type botulinum neurotoxin, preparation method and use thereof

A botulinum neurotoxin and monoclonal antibody technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of limited therapeutic effect, achieve high affinity, good application prospects, and inhibit neurotoxicity. effect of effect

Active Publication Date: 2012-05-30
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, research-related reports have shown that single antibody clones have limited therapeutic effect on botulinum toxin poisoning, thus prompting people to search for more and more effective monoclonal antibodies to develop a monoclonal antibody cocktail treatment strategy

Method used

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  • Neutralizing monoclonal antibodies against B type botulinum neurotoxin, preparation method and use thereof
  • Neutralizing monoclonal antibodies against B type botulinum neurotoxin, preparation method and use thereof
  • Neutralizing monoclonal antibodies against B type botulinum neurotoxin, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction and screening of mouse-derived anti-botulinum neurotoxin type B phage immune library

[0027] 1. Materials:

[0028] 1. Bacterial strains: the helper phage M13K07 was purchased from Biolab Company; the host strain E.coli TG1 was a product of Pharmacia Company.

[0029] 2. Reagents: NotI, SfiI and other restriction enzymes are all products of Promega; FicollPlus Pague and anti M13-HRP are products of Pharmacia; total RNA extraction kit, RT reverse transcription kit, PCR amplification reagent are products of TAKARA company ; BSA (bovine serum albumin fraction V) is a product of GIBCO; DEPC, carbenicillin, tetracycline and kanamycin sulfate were purchased from SIGMA.

[0030] 3. Vector: The phage vector pCANTAB-5E is a product of Pharmacia.

[0031] 2. Method results:

[0032] 1. Recombinant preparation of type B botulinum toxin receptor binding region Hc protein (BHc) and animal immunization

[0033] 1.1 Recombinant Production of Type B Botulinu...

Embodiment 2

[0057] Example 2 Construction of humanized anti-botulinum neurotoxin type B antibody clone

[0058] 1. Materials:

[0059] 1. Strains: the host strain E.coli DH5a is a product of Novagen.

[0060] 2. Reagents: PCR amplification reagents are products of TAKARA Company.

[0061] 3. Carrier: The pMD18-T carrier is a product of TaKaRa Company,

[0062] 2. Method results:

[0063] 1. MuBNbP8 gene mutation

[0064] Use the Blast search database in GenBank to input the MuBNbP8 antibody sequence and human antibody for homologous alignment to determine the conserved residues. At the same time, computer-aided design is used to determine the humanized transformation site of the murine antibody. The principle is that the modified residues are located on the surface of the antibody molecule, and the residue replacement does not affect the space where the antibody binds to the antigen. According to homologous comparison and computer-aided design, the mutation sites were determined as: ...

Embodiment 3

[0067] Example 3 Recombinant Preparation of Mouse and Humanized Anti-botulinum Toxin Type B Genetic Engineering Antibodies

[0068] 1. Materials:

[0069] 1. Strains: host bacteria E.coli ER2566, TB1, DH5α are products of New England Biolab (NEB); E.coli BL21 (DE3) are products of Novagen.

[0070] 2. Reagents: Amylose affinity chromatography column, anti-MBP enzyme-labeled monoclonal antibody were purchased from NEB Company; PCR amplification reagents were products from TAKARA Company; plasmid mini-extraction kit was purchased from Tiangen Company, T4 ligase, restriction endogenous Dicer is a product of NEB Company; DL2000, DNA molecular weight standards and medium and low molecular weight protein standards are products of Tiangen Company; carbenicillin was purchased from SIGMA Company.

[0071] 3. Vector: The recombinant expression vector pMAL-C2X is a product of NEB Company.

[0072] 2. Method results:

[0073] 1. Recombinant preparation of MuBNbP8 and HuBNbP8

[0074] ...

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Abstract

The invention discloses a neutrality monoclonal antibody against botulinum neurotoxin type B and also discloses a humanized modifier of the antibody. The antibody is prepared through the following method: firstly, the CDR region, variable region, IgG part or full gene of the antibody is prepared through molecular biological methods or other methods and is expressed in prokaryotic cells such as colibacillus, eukaryote cells such as yeast, or insect cells, vegetable cells or mammal cells such as CHO; and the antibody is obtained through purification. The antibody is characterized by strong specific neutralization activity and high affinity. In addition, the humanized antibody is characterized by low side and toxic effect. The neutrality monoclonal antibody and the humanized modifier of the antibody are suitable for the first aid treatment of botulinum neurotoxin type B intoxication and also for the detection of botulinum neurotoxin type B. Therefore, the antibody and the humanized modifier thereof have good application prospect.

Description

technical field [0001] The invention relates to an anti-toxin monoclonal antibody, in particular to an anti-botulinum neurotoxin neutralizing monoclonal antibody, and also relates to a preparation method and application of the antibody. Background technique [0002] Clostridium Botulinum Neurotoxins (Clostridium Botulinum Neurotoxins, BN, referred to as botulinum toxin) is a group of known most virulent proteins (including A-G types). When botulinum toxin enters the human circulation from contaminated food or infection, it causes severe neurological disorders and progressive neuromuscular paralysis by blocking the release of the neurotransmitter acetylcholine (excitatory synaptic transmitter) at the neuromuscular junction. Poisoning symptoms, resulting in skeletal muscle weakness and bulbar paralysis, severe respiratory failure, cardiac arrest and death. The main causes of human poisoning are A, B, and E types. Case statistics show that type B botulinum toxin is the most c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/12C12N15/63A61K39/40G01N33/577A61P31/04
CPCY02A50/30
Inventor 王慧史晶荫俊侯晓军蔡昆包士中王琴
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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