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Human source anti-A botulinum neurotoxin genetic engineering antibody and preparation method and use thereof

A technology of botulinum neurotoxin and genetically engineered antibody, which is applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as poor stability, achieve high affinity, good application prospects, and eliminate allergic reactions Effect

Active Publication Date: 2009-12-30
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

[0008] Aiming at the problem of poor stability that usually exists in single-chain antibodies, the present invention also provides a small molecule stability variant (dsFv) of a human-derived anti-botulinum neurotoxin type A genetically engineered antibody, i.e. disulfide bond stability Fvs

Method used

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  • Human source anti-A botulinum neurotoxin genetic engineering antibody and preparation method and use thereof
  • Human source anti-A botulinum neurotoxin genetic engineering antibody and preparation method and use thereof
  • Human source anti-A botulinum neurotoxin genetic engineering antibody and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction and Screening of Human Anti-Botulinum Toxin Type A Phage Immune Library

[0033] 1. Materials:

[0034] 1. Bacterial strains: the helper phage M13K07 was purchased from Biolab Company; the host strain E.coli TG1 was a product of Pharmacia Company.

[0035] 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.

[0036] 3. Vector: The phage vector pCANTAB-5E is a product of Pharmacia.

[0037] 2. Method results:

[0038] 1. Type A botulinum toxin epitope peptide vaccine and volunteer immunization

[0039] 1.1 Type A botulinum toxin epitope peptide synthesis

[0040] Peptide A (BoNTaHc1157-1181, Pa): as...

Embodiment 2

[0077] Example 2 Recombinant Preparation of Human Anti-Botulinum Toxin Type A Genetic Engineering Antibody

[0078] 1. Materials:

[0079] 1. Strains: the host strain E.coli HB2151 is a product of Pharmacia; E.coli BL21(DE3) is a product of Novagen.

[0080] 2. Reagents: E-tag affinity chromatography purification medium and Histrap HP prepacked column for Ni-NTA affinity purification were purchased from Amersham Pharmacia; PCR amplification reagents were products from TAKARA; carbenicillin was purchased from SIGMA; imidazole Purchased from Amresco Corporation.

[0081] 3. Vector: The phage recombinant expression vector pCANTAB-5E is a product of Pharmacia; the expression vector pET22b is a product of Novagen.

[0082] 2. Method results:

[0083] 1. Recombinant preparation of ScFv

[0084] 1.1 Soluble expression of ScFv in Escherichia coli

[0085] The recombinant expression plasmid pCan-ScFv and the empty vector of the positive clone obtained by screening were transformed...

Embodiment 3

[0100] Example 3 Analysis of Biological Activity of Human Anti-Botulinum Toxin Type A Genetic Engineering Antibody

[0101] 1. Materials:

[0102] 1. Reagents: anti His-HRP is the product of Pierce Company; BSA (Bovine Serum Albumin Fraction V) is the product of GIBCO Company; ammonium thiocyanate was purchased from Beijing Biochemical Reagent Company; anti-botulinum horse serum was purchased from China Pharmaceutical Biological Products Laboratory; Reagents for affinity determination such as HBS solution, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N hydroxysuccinimide (NHS), and ethanolamine are all from Pharmacia Company product.

[0103] 2. Materials: CM5 chips are products of Pharmacia; Balb / C mice were purchased from the Experimental Animal Center of Academy of Military Medical Sciences.

[0104] 3. Instrument: Biacore3000 is a product of GE.

[0105] 2. Method results:

[0106] 1. Antigen-binding activity (ELISA) of human-derived anti-botulinum toxin type ...

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Abstract

This invention relates to human source, a type kreotoxin resistant neurotoxin monoclonal antibody. This antibody possesses amino acid sequence as 35 in sequence table. This invention also discloses isomeride of above antibody, its amino acid sequence of heavy chain as 36 in sequence table, amino acid sequence of light chain as 37 in sequence table. Above antibody through following method preparing: first by molecular biology or other method prepare HuAb - BNa gene that possess idiosyncratic neutralize activity and high affinity, express in prokaryotic cell such as colibacillus, eukaryon as microzyme, mammal cell as CHO and so on, after purification to obatin homogeneous HuAb - BNa protein. This HuAb - BNa possess trait of strong idiosyncratic neutralize activity, high affinity, and no side effect, lend itself to first-aid treatment of A type kreotoxism neurotoxin, at the same time lend itself to detect of A type kreotoxism neurotoxin, possess favorable application prospects.

Description

technical field [0001] The invention relates to a genetically engineered antibody, in particular to a human-sourced anti-botulinum neurotoxin genetically engineered antibody, and also relates to a preparation method and application of the antibody. Background technique [0002] Botulinum neurotoxin (Clostridium Botulihum Neurotoxins, BN, referred to as botulinum toxin) is a group of known most virulent proteins (including A-G type). 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. Type A, B, and E are the main causes of human poisoning, and type A botulinum toxin can cause more serious symptoms and lea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12P21/00A61K39/395A61P39/02G01N33/53
CPCY02A50/30
Inventor 王慧荫俊史晶侯晓军包士中王忠泽蔡昆
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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